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PubMed2002

Ratiometric probes for hydrogencarbonate analysis in intracellular or extracellular environments using europium luminescence.

A series of six, cationic, zwitterionic and anionic Eu complexes has been examined for the analysis of hydrogencarbonate concentration in the intracellular and extracellular ranges; an anionic complex incorporating three glutarate residues and a sensitising acridone chromophore (lambda exc = 410 nm) exhibits a 69% change in the intensity ratio of the 618/588 nm Eu emission bands between 5 and 15 mM HCO3- in a cell lysate medium.

Journal Article↗
PubMed2004

Synthesis and photophysical evaluation of charge neutral thiourea or urea based fluorescent PET sensors for bis-carboxylates and pyrophosphate.

The synthesis of the fluorescent photoinduced electron transfer (PET) chemosensors 1-3 for bis-anions such as bis-carboxylates and pyrophosphate in organic solvents is described herein. These sensors are based on the receptor-spacer-fluorophore-spacer-receptor motif where the receptors are charge neutral aromatic thiourea or urea receptors and the fluorophore is anthracene. The anion recognition was evaluated using 1H NMR as well as absorption and fluorescence spectroscopy in DMSO. For simple anions such as acetate or fluoride, the recognition was shown to be through hydrogen bonding of the corresponding anion to the receptors. This gave rise to only minor changes in the absorption spectra, but significant changes in the fluorescence emission spectra, which was substantially (70-95%) quenched. Analysis of these recognition events implied a 1 : 2 (sensor : anion) binding and ideal PET behaviour for ions such as AcO- and H2PO4-. For F-, the luminescent quenching indicated a 1 : 1 binding, but we deduced that this was due more to complete quenching of the excited state after the addition of one equivalent of the anion. For all of the anions, the quenching contributed to enhanced efficiency of PET from the receptors to the excited state of the fluorophore. In the case of the bis-anions (ambient), such as di-carboxylates, similar fluorescence quenching was observed. However, here either a 1 : 1 or a 1 : 2 binding was observed depending on the length of the spacer separating the two carboxylate moieties and the nature of the receptor. Whereas both pyrophosphate and malonate gave rise to a 1 : 1 binding, glutarate gave rise to approximately 1 : 2 binding for the thiourea sensors 1 and 2. However, for the urea based sensor 3, the binding was found to be 1 : 1 for all the bis-anions. For such a 1 : 1 binding we propose that the anion most likely bridges the fluorophore moiety. This was also evident from the 1H NMR (DMSO-d6) spectrum where the anthracene resonances were significantly affected. By simply modifying the electronic structure of the receptor, the sensitivity of the recognition process could also be modified; e.g. compound 1, bearing the trifluoromethyl substituent, showed stronger binding to the bis-anions than 2, which possessed a simple phenyl moiety.

Chemical Phenomena↗
PubMed1965

EXCHANGE TRANSAMINATION AND THE METABOLISM OF GLUTAMATE IN BRAIN.

1. Experiments were performed to throw light on why the incorporation of (14)C from labelled carbohydrate precursors into glutamate has been found to be more marked in brain than in other tissues. 2. Rapid isotope exchange between labelled glutamate and unlabelled alpha-oxoglutarate was demonstrated in brain and liver mitochondrial preparations. In the presence but not in the absence of alpha-oxoglutarate the yield of (14)CO(2) from [1-(14)C]glutamate exceeded the net glutamate removal, and the final relative specific activities of the two substrates indicated that complete isotopic equilibration had occurred. Also, when in a brain preparation net glutamate removal was inhibited by malonate, isotope exchange between [1-(14)C]glutamate and alpha-oxoglutarate and the formation of (14)CO(2) were unaffected. 3. The time-course of isotope exchange between labelled glutamate and unlabelled alpha-oxoglutarate was followed in uncoupled brain and liver mitochondrial fractions, and the rate of exchange calculated by a computer was found to be 3-8 times more rapid than the maximal rate of utilization of the two substrates. 4. The physiological situation was imitated by the continuous infusion of small amounts of alpha-oxo[1-(14)C]glutarate into brain homogenate containing added glutamate. The fraction of (14)C infused that was retained in the glutamate pool depended on the size of the latter, and the final relative specific activities of the two substrates indicated almost complete isotope exchange. Isotopic equilibration also occurred when alpha-oxoglutarate was generated from pyruvate through the tricarboxylic acid cycle in a brain mitochondrial preparation containing [1-(14)C]glutamate. 5. The differences in the incorporation of (14)C from labelled glucose into the glutamate of brain and liver are discussed in terms of the rates of isotope exchange, the glutamate pool sizes and the rates of formation of labelled alpha-oxoglutarate in the two tissues. It is concluded that the differences between tissues in the incorporation of glucose carbon into glutamate reflect features of their metabolism largely unrelated to that of glutamate.

Amino Acids↗
PubMed1965

Studies on the biosynthesis of biotin. Production of biotin and biotin-like compounds by a pseudomonad.

1. Filtrates from cultures of a strain of Pseudomonas aeruginosa, grown in a basal glucose-ammonium chloride-vitamins-salts medium, possessed biotin activity as detected by microbiological assays. Exponential-phase culture filtrates contained biotin and desthiobiotin in the approximate ratio 1:3, with smaller amounts of biotin sulphoxide and three unidentified compounds with biotin activity. 2. The addition of malonate, adipate or pimelate to the basal medium stimulated the production of compounds with biotin activity; this effect was enhanced when these compounds were included in the medium as the major carbon source. Succinate, glutarate, suberate, fumarate or oxaloacetate did not stimulate the production of compounds with biotin activity. The ratio of biotin to desthiobiotin in filtrates from cultures grown in medium containing malonate as the carbon source was about 1:1. Experiments in which mixtures of malonate and pimelate were included in the medium as the carbon sources showed that these acids probably make a similar contribution in biotin biosynthesis. 3. A number of heterocyclic compounds, including several containing the ureido group (-NH-CO-NH-), were included in the basal medium but none of them stimulated the production of compounds with biotin activity to any marked degree. 4. Several amino acids, particularly cysteine (or cystine) and lysine, when added individually as supplements to the basal medium, stimulated the production of compounds with biotin activity. Filtrates from cultures grown in medium supplemented with cysteine contained approximately equal proportions of biotin and desthiobiotin. A much greater stimulation in the production of compounds with biotin activity was obtained when certain amino acids were included in the medium as the major source of nitrogen or carbon and nitrogen; ornithine, citrulline and argininosuccinate had the most marked effect. The ratio of biotin to desthiobiotin in filtrates from these cultures was usually greater than in filtrates from cultures grown in basal medium. 5-Aminovalerate also caused some stimulation when used as the nitrogen source, but urea was inactive. The effect of binary mixtures of certain amino acids was also examined. 5. The results are discussed in relation to the possible role of the stimulatory compounds during biotin biosynthesis.

Amino Acids↗
PubMed1967

The synergistic decarboxylation of glyoxylate and 2-oxoglutarate by an enzyme system from pig-liver mitochondria.

1. An enzyme system that catalyses a synergistic decarboxylation of glyoxylate and 2-oxoglutarate has been purified from pig-liver mitochondria. 2. The purified system is specific for glyoxylate and 2-oxoglutarate as substrates, although in earlier stages of purification glycine and l-glutamate are also active. 3. The reaction is inhibited strongly by EDTA and N-ethylmaleimide. Substrate analogues, present at concentrations equimolar with respect to the substrates, are not effective as inhibitors. 4. The reaction proceeds in the absence of added cofactors. Magnesium chloride, mercaptoethanol and sucrose stimulate the reaction, and stabilize the activity of the enzyme. 5. The pH optimum of the reaction is 7.0. The K(m) values of glyoxylate and 2-oxoglutarate, at saturating concentration of the corresponding co-substrate, are 16mm and 3.6mm respectively. 6. Isotopic work with specifically labelled [(14)C]glyoxylate and 2-oxo[(14)C]-glutarate suggests that the enzyme system catalyses an initial condensation of glyoxylate and 2-oxoglutarate that results in, or leads to, release of C-1 of both substrates as carbon dioxide. C-2 of glyoxylate and C-5 of 2-oxoglutarate do not appear as carbon dioxide. 7. The stoicheiometry of the reaction is complex. During the initial stages of the reaction, more carbon dioxide is recovered from 2-oxoglutarate than from glyoxylate. Subsequently, there is a disproportionate increase with time of carbon dioxide evolution from the carboxyl group of glyoxylate. The excess of decarboxylation of glyoxylate over 2-oxogluturate is further increased by treatment of reaction products with acid.

Journal Article↗
PubMed1967

The fate of[14C]thalidomide in the pregnant rabbit.

1. The fate of [(14)C]thalidomide orally administered to pregnant rabbits at the beginning of the sensitive phase of pregnancy has been studied. 2. After the oral administration of [(14)C]thalidomide on the 192nd hour of pregnancy about 68% of the radioactivity appears in the urine and 22% in the faeces. 3. The urinary (14)C is made up as follows (% of dose): thalidomide (2); alpha-(o-carboxybenzamido)glutarimide (16); 2- and 4-phthalimidoglutaramic acids (11); 2-phthalimidoglutaric acid (0.2); 2- and 4-(o-carboxybenzamido)glutaramic acids and 2-(o-carboxybenzamido)-glutaric acid (29). 4. The plasma (14)C concentration is maximal at 12hr. after dosing and the radioactivity persists for more than 58hr. At 4hr. the main compound in the plasma is thalidomide, but its concentration steadily declines while the concentration of its hydrolysis products increases. 5. At 12, 24 and 58hr. after dosing radioactivity is present in the embryo and the maternal tissues examined. The (14)C concentration in the embryo is at nearly all times higher than that in the plasma, brain, skeletal muscle and fat but lower than that in the liver and kidney. 6. At 4hr. after dosing the mother on the tenth day of pregnancy the specific activities of the embryo and the yolk-sac fluid are similar. 7. Thalidomide is found in the embryo together with seven of its hydrolysis products for more than 24hr. after dosing. The accumulation of radioactivity in the embryo is due to retention of the polar hydrolysis products.

Adipose Tissue↗
PubMed1970

Gluconeogenesis in the cow. The effects of a glucocorticoid on hepatic intermediary metabolism.

1. The hepatic concentrations of the ketone bodies and of metabolites and activities of enzymes involved in gluconeogenesis were measured in healthy lactating and non-lactating cows 48h after administration of Voren, an ester of dexamethasone, and compared with those found in control animals given saline. Parallel measurements were also made of the blood concentrations of several of the metabolites. 2. Blood glucose concentrations were raised in the Voren-treated animals, whereas blood ketone body and free fatty acid concentrations were unaltered. Similarly there was no change in the hepatic concentrations of the ketone bodies. 3. Significant increases were found in the hepatic concentrations of citrate, 2-oxo-glutarate and malate in both groups of animals given Voren. 4. The hepatic concentrations of those glycolytic intermediates that were measured either decreased or did not change after Voren treatment. 5. The enzymes aspartate transaminase and fructose 1,6-diphosphatase were unchanged in activity after Voren administration, whereas phosphopyruvate carboxylase (EC 4.1.1.32) activity was depressed in the lactating group. However, glucose 6-phosphatase, tryptophan oxygenase and tyrosine aminotransferase increased in activity. 6. In several cases those hepatic metabolites that increased in concentration after Voren administration were present in lower concentration in normal lactating cows than in normal non-lactating cows. The same applied mutatis mutandis to those metabolites that were decreased by Voren. 7. These findings are discussed in relation to the use of glucocorticoids in the treatment of bovine ketosis.

Amino Acids↗
PubMed1974

Inhibition by the branched-chain 2-oxo acids of the 2-oxoglutarate dehydrogenase complex in developing rat and human brain.

1. The effect of the branched-chain amino acids, namely leucine, isoleucine and valine and their corresponding 2-oxo acids on the metabolism of 2-oxoglutarate by developing rat and human brain preparations was investigated. 2. The decarboxylation of 2-oxo[1-(14)C]glutarate to (14)CO(2) by mitochondria from adult rat brain was inhibited by the branched-chain 2-oxo acids whereas the branched-chain amino acids had no inhibitory effect on this process. 3. The activity of 2-oxoglutarate dehydrogenase complex was about 0.2unit/g of brain from 2-day-old rats and increased by about fourfold reaching an adult value by the end of the third postnatal week. 4. The K(m) value for 2-oxoglutarate of the 2-oxoglutarate dehydrogenase complex in rat and human brain was 100 and 83mum respectively. 5. The branched-chain 2-oxo acids competitively inhibited this enzyme from suckling and adult rats brains as well as from foetal and adult human brains, whereas the branched-chain amino acids had no effect on this enzyme. 6. Approximate K(i) values for the branched-chain 2-oxo acids found for this enzyme were in the range found for these 2-oxo acids in plasma from patients with maple-syrup-urine disease. 7. The possible significance of the inhibition by the branched-chain 2-oxo acids of the 2-oxoglutarate dehydrogenase complex in brains of untreated patients with maple-syrup-urine disease is discussed in relation to the energy metabolism and the biosynthesis of lipids from ketone bodies.

Age Factors↗
PubMed1976

Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide.

The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.

Acetates↗
PubMed1978

Inter-organ relationships between glucose, lactate and amino acids in rats fed on high-carbohydrate or high-protein diets.

1. Inter-organ relationships between glucose, lactate and amino acids were studied by determination of plasma concentrations in different blood vessels of anaesthetized rats fed on either a high-carbohydrate diet [13% (w/w) casein, 79% (w/w) starch] or a high-protein diet [50% (w/w) casein, 42% (w/w) starch]. The period of food intake was limited (09:00-17:00h), and blood was collected 4h after the start of this period (13:00h). 2. Glucose absorption was considerable only in rats fed on a high-carbohydrate diet. Portal-vein-artery differences in plasma lactate concentration were higher in rats fed on this diet, but not proportional to glucose absorption. Aspartate, glutamate and glutamine were apparently converted into alanine, but when dietary protein intake was high, a net absorption of glutamine occurred. 3. The liver removed glucose from the blood in rats fed on a high-carbohydrate diet, but glucose was released into the blood in rats fed on the high-protein diet, probably as a result of gluconeogenesis. Lactate uptake was very low when amino acid availability was high. 4. In rats on a high-protein diet, increased uptake of amino acids, except for ornithine, was associated with a rise in portal-vein plasma concentrations, and in many cases with a decrease in hepatic concentrations. 5. Hepatic concentrations of pyruvate and 2-oxo-glutarate decreased without a concomitant change in the concentrations of lactate and malate in rats fed on the high-protein diet, in spite of an increased supply of pyruvate precursors (e.g. alanine, serine, glycine), suggesting increased pyruvate transport into mitochondria. 6. High postprandial concentrations of plasma glucose and lactate resulted in high uptakes of these metabolites in peripheral tissues of rats on both diets. Glutamine was released peripherally in both cases, whereas alanine was taken up in rats fed on a high-carbohydrate diet, but released when the amino acid supply increased. 7. It is concluded that: the small intestine is the main site of lactate production, and the peripheral tissues are the main site for lactate utilization; during increased ureogenesis in fed rats, lactate is poorly utilized by the liver; the gut is the main site of alanine production in rats fed on a high-carbohydrate diet and the liver utilizes most of the alanine introduced into the portal-vein plasma in both cases.

Amino Acids↗
PubMed1979

Kinetic studies of dogfish liver glutamate dehydrogenase.

Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621--631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver--Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5'-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free enzyme.

Ammonia↗
PubMed1981

The metabolism of L-tryptophan by liver cells prepared from adrenalectomized and streptozotocin-diabetic rats.

1. The metabolism of L-tryptophan by liver cells prepared from fed normal, adrenalectomized and streptozotocin-diabetic rats was studied. 2. At physiological concentrations (0.1 mM), the rate of oxidation of tryptophan by tryptophan 2,3-dioxygenase was 3-fold greater in liver cells from diabetic rats than in those from fed rats. In liver cells from diabetic rats, oxidation of tryptophan to CO2 and metabolites of the glutarate pathway was increased 7-fold. Quinolinate synthesis was decreased by 50%. These findings are consistent with an increase in picolinate carboxylase activity. 3. Rates of metabolism of 0.1 mM-tryptophan by hepatocytes from fed and adrenalectomized rats were similar. 4. In all three types of cell preparation, fluxes through tryptophan 2,3-dioxygenase with 2.5 mM-tryptophan were 7-fold greater than those obtained with 0.1 mM-tryptophan. Tryptophan 2,3-dioxygenase and kynureninase fluxes in hepatocytes from fed and adrenalectomized rats were comparable, whereas those in liver cells from diabetic rats were increased 2.5-fold and 3.3-fold respectively. Picolinate carboxylase activities of liver cells from diabetic rats were 15-fold greater than those of cells from fed rats, but rates of quinolinate synthesis were unchanged. 5. It is concluded that: (i) adrenal corticosteroids are not required for the maintenance of basal activities of the kynurenine pathway, whereas (ii) chronic insulin deficiency produces changes in both the rate of oxidation and metabolic fate of tryptophan carbon.

Acetyl Coenzyme A↗
PubMed2004

Functional features and genomic organization of mouse NaCT, a sodium-coupled transporter for tricarboxylic acid cycle intermediates.

In the present study, we report on the molecular cloning and functional characterization of mouse NaCT (Na+-coupled citrate transporter), the mouse orthologue of Drosophila Indy. Mouse NaCT consists of 572 amino acids and is highly similar to rat and human NaCTs in primary sequence. The mouse nact gene coding for the transporter is approx. 23 kb long and consists of 12 exons. When expressed in mammalian cells, the cloned transporter mediates the Na+-coupled transport of citrate and succinate. Competition experiments reveal that mouse NaCT also recognizes other tricarboxylic acid cycle intermediates such as malate, fumarate and 2-oxo-glutarate as excellent substrates. The Michaelis-Menten constant for the transport process is 38+/-5 mM for citrate and 37+/-6 mM for succinate at pH 7.5. The transport process is electrogenic and exhibits an obligatory requirement for Na+. Na+-activation kinetics indicates that multiple Na+ ions are involved in the activation process. Extracellular pH has a differential effect on the transport function of mouse NaCT depending on whether the transported substrate is citrate or succinate. The Michaelis-Menten constants for these substrates are also influenced markedly by pH. When examined in the Xenopus laevis oocyte expression system with the two-microelectrode voltage-clamp technique, the transport process mediated by mouse NaCT is electrogenic. The charge-to-substrate ratio is 1 for citrate and 2 for succinate. The most probable transport mechanism predicted by these studies involves the transport of citrate as a tervalent anion and succinate as a bivalent anion with a fixed Na+/substrate stoichiometry of 4:1. The present study provides the first unequivocal evidence for the electrogenic nature of mammalian NaCT.

Amino Acid Sequence↗
PubMed2006

Oxo-4-methylpentanoic acid directs the metabolism of GABA into the Krebs cycle in rat pancreatic islets.

OMP (oxo-4-methylpentanoic acid) stimulates by itself a biphasic secretion of insulin whereas L-leucine requires the presence of L-glutamine. L-Glutamine is predominantly converted into GABA (gamma-aminobutyric acid) in rat islets and L-leucine seems to promote its metabolism in the 'GABA shunt' [Fernández-Pascual, Mukala-Nsengu-Tshibangu, Martín del Río and Tamarit-Rodríguez (2004) Biochem. J. 379, 721-729]. In the present study, we have investigated how 10 mM OMP affects L-glutamine metabolism to uncover possible differences with L-leucine that might help to elucidate whether they share a common mechanism of stimulation of insulin secretion. In contrast with L-leucine, OMP alone stimulated a biphasic insulin secretion in rat perifused islets and decreased the islet content of GABA without modifying its extracellular release irrespective of the concentration of L-glutamine in the medium. GABA was transaminated to L-leucine whose intracellular concentration did not change because it was efficiently transported out of the islet cells. The L-[U-14C]-Glutamine (at 0.5 and 10.0 mM) conversion to 14CO2 was enhanced by 10 mM OMP within 30% and 70% respectively. Gabaculine (250 microM), a GABA transaminase inhibitor, suppressed OMP-induced oxygen consumption but not L-leucine- or glucose-stimulated respiration. It also suppressed the OMP-induced decrease in islet GABA content and the OMP-induced increase in insulin release. These results support the view that OMP promotes islet metabolism in the 'GABA shunt' generating 2-oxo-glutarate, in the branched-chain alpha-amino acid transaminase reaction, which would in turn trigger GABA deamination by GABA transaminase. OMP, but not L-leucine, suppressed islet semialdehyde succinic acid reductase activity and this might shift the metabolic flux of the 'GABA shunt' from gamma-hydroxybutyrate to succinic acid production.

Aldehyde Oxidoreductases↗
PubMed1983

Stimulation by alpha-adrenergic agonists of Ca2+ fluxes, mitochondrial oxidation and gluconeogenesis in perfused rat liver.

Glucose output from perfused livers of 48 h-starved rats was stimulated by phenylephrine (2 microM) when lactate, pyruvate, alanine, glycerol, sorbitol, dihydroxyacetone or fructose were used as gluconeogenic precursors. Phenylephrine-induced increases in glucose output were immediately preceded by a transient efflux of Ca2+ and a sustained increase in oxygen uptake. Phenylephrine decreased the perfusate [lactate]/[pyruvate] ratio when sorbitol or glycerol was present, but increased the ratio when alanine, dihydroxyacetone or fructose was present. Phenylephrine induced a rapid increase in the perfusate [beta-hydroxybutyrate]/[acetoacetate] ratio and increased total ketone-body output by 40-50% with all substrates. The oxidation of [1-14C]octanoate or 2-oxo[1-14C]glutarate to 14CO2 was increased by up to 200% by phenylephrine. All responses to phenylephrine infusion were diminished after depletion of the hepatic alpha-agonist-sensitive pool of Ca2+ and returned toward maximal responses after Ca2+ re-addition. Phenylephrine-induced increases in glucose output from lactate, sorbitol and glycerol were inhibited by the transaminase inhibitor amino-oxyacetate by 95%, 75% and 66% respectively. Data presented suggest that the mobilization of an intracellular pool of Ca2+ is involved in the activation of gluconeogenesis by alpha-adrenergic agonists in perfused rat liver. alpha-Adrenergic activation of gluconeogenesis is apparently accompanied by increases in fatty acid oxidation and tricarboxylic acid-cycle flux. An enhanced transfer of reducing equivalents from the cytoplasmic to the mitochondrial compartment may also be involved in the stimulation of glucose output from the relatively reduced substrates glycerol and sorbitol and may arise principally from an increased flux through the malate-aspartate shuttle.

Animals↗
PubMed1983

Tryptophan and glucose metabolism in rat liver cells. The effects of DL-6-chlorotryptophan, 4-chloro-3-hydroxyanthranilate and pyrazinamide.

Liver cells pre-incubated with 1 mM-DL-6-chlorotryptophan are less sensitive to tryptophan-mediated inhibition of gluconeogenesis; this effect is apparent both at physiological (0.1 mM) and higher (0.5 mM) concentrations of tryptophan. 4-Chloro-3-hydroxyanthranilate (1-100 microM) has effects similar to those of DL-6-chlorotryptophan. The effects of both compounds are consistent with a decrease in quinolinate formation, a consequence of inhibition of 3-hydroxyanthranilate oxidase. Pyrazinamide (0.25-5.0 mM) significantly decreased flux through the glutarate pathway and potentiated tryptophan-mediated inhibition of gluconeogenesis; these changes were apparent at physiological concentrations of tryptophan. The effects of pyrazinamide are consistent with an increase in quinolinate formation resulting from inhibition of picolinate carboxylase.

3-Hydroxyanthranilic Acid↗
PubMed1985

The role of tryptophan 2,3-dioxygenase in the hormonal control of tryptophan metabolism in isolated rat liver cells. Effects of glucocorticoids and experimental diabetes.

The metabolism of L-tryptophan by isolated liver cells prepared from control, adrenalectomized, glucocorticoid-treated, acute-diabetic, chronic-diabetic and insulin-treated chronic-diabetic rats was studied. Liver cells from adrenalectomized rats metabolized tryptophan at rates comparable with the minimum diurnal rates of controls, but different from rates determined for cells from control rats 4h later. Administration of dexamethasone phosphate increased the activity of tryptophan 2,3-dioxygenase (EC 1.13.11.11) 7-8-fold, and the flux through the kynurenine pathway 3-4-fold, in cells from both control and adrenalectomized rats. Increases in flux through kynureninase (EC 3.7.1.3) and to acetyl-CoA can be explained in terms of increased substrate supply from tryptophan 2,3-dioxygenase. The metabolism of tryptophan was increased 3-fold in liver cells isolated from acutely (3 days) diabetic rats, with a 7-8-fold increase in the maximal activity of tryptophan 2,3-dioxygenase. The oxidation of tryptophan to CO2 and metabolites of the glutarate pathway increased 4-5-fold, consistent with an increase in picolinate carboxylase (EC 4.1.1.45) activity. Liver cells isolated from chronic (10 days) diabetic rats metabolized tryptophan at rates comparable with those of cells from acutely diabetic rats, but with a 50% decrease in the activity of tryptophan 2,3-dioxygenase. The proportion of flux from tryptophan 2,3-dioxygenase to acetyl-CoA, however, was increased by 50%; this was indicative of further increases in the activity of picolinate carboxylase. Administration of insulin partially reversed the effects of chronic diabetes on the activity of tryptophan 2,3-dioxygenase and flux through the kynurenine pathway, but had no effect on the increased activity of picolinate carboxylase. The role of tryptophan 2,3-dioxygenase in regulating the blood tryptophan concentration is discussed with reference to its sensitivity to the above conditions.

Adrenalectomy↗
PubMed1985

Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria.

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent 'State 3.5' respiration condition. Ca2+ had no effect on NAD(P)H formation induced by beta-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed.

Animals↗