Effect of aminooxyacetate and α-ketoglutarate on glutamate deamination by rat kidney mitochondria

• 1.1. Glutamate dehydrogenase flux by rat kidney mitochondria incubated with 1 mM glutamine plus 2–3 mM glutamate was stimulated by aminooxyacetate. This effect was inhibited by α-ketoglutarate.
• 2.2. Studies with intact mitochondria and mitochondrial sonicates revealed a linear inverse relationship between glutamate deamination and α-ketoglutarate levels.
• 3.3. The data revealed that α-ketoglutarate is a competitive inhibitor of glutamate dehydrogenase with an apparent K_i of 0.6mM.
• 4.4. The data suggest that aminooxyacetate stimulates glutamate deamination by a mechanism mediated by α-ketoglutarate.

     

Effect of adenosine on gluconeogenesis in the perfused liver of chicken and guinea-pig

• 1.1. Glucose formation from lactate by the perfused liver of 48 hr starved chickens was strongly inhibited by adenosine (Ado); the half-maximal inhibition was attained at 40 μM. This effect was paralleled by a four- to five-fold increase of ATP content as determined in freeze-clamped liver.
• 2.2. In chicken liver homogenate gluconeogenesis from precursors such as alanine, glutamate, glutamine and aspartate, which are not converted into glucose by the perfused chicken liver, proceeded at rates equal to or higher than that with lactate, being markedly inhibited by Ado.
• 3.3. In the perfused guinea-pig liver glucose synthesis with lactate, propionate, glycerol and fructose was also inhibited by Ado; however, when precursors such as pyruvate, glutamine and a mixture of lactate + pyruvate were supplied to the liver Ado did not inhibit gluconeogenesis.
• 4.4. Assay of adenine nucleotides in the perfused guinea-pig liver, stopped by freeze-clamping technique in a number of experimental variants, revealed no correlation between the rate of gluconeogenesis and the changes induced by Ado in the adenine nucleotide pool.
• 5.5. In the perfused liver of both chicken and guinea-pig Ado produced an increase of the lactate to pyruvate ratio and, in general, a diminution of the content of malate-aspartate shuttle intermediates.
• 6.6. The results are interpreted as suggesting that the inhibitory effect of Ado on hepatic gluconeogenesis is not necessarily mediated by the changes in the adenine nucleotide pool.

     

Interrelationships between gluconeogenesis and uricogenesis in chicken liver

• 1.1. Experiments performed on isolated hepatocytes and perfused liver of starved chickens showed that gluconeogenesis from lactate, glycerol and fructose was inhibited by 22–100% on addition of urate precursors.
• 2.2. The inhibition was associated with an increased rate of urate formation.
• 3.3. 2,4-Dinitrophenol (40 μM), 2-bromooctanoate (2 mM) and 3-mercaptopicolinate (3MPA) (0.5 mM) were inhibitory with respect to gluconeogenesis but did not significantly affect the rate of urate formation.
• 4.4. The possible interrelationships between gluconeogenesis and uricogenesis are considered in terms of a competition for ATP and for other metabolites between the two pathways.
• 5.5. An interplay of both pathways at the level of anion transfer across the inner mitochondrial membrane is also discussed.

     

Effects of the nonsteroidal anti-inflammatory drug piroxicam on energy metabolism in the perfused rat liver

• 1.1. The actions of piroxicam, a nonsteroidal and noncarboxylic anti-inflammatory drug, on the metabolism of the isolated perfused rat liver were investigated. The main purpose was to verify if piroxicam is also active on glycogenolysis and energy metabolism, as demonstrated for several carboxylic nonsteroidal anti-inflammatories.
• 2.2. Piroxicam increased oxygen consumption in livers from both fed and fasted rats.
• 3.3. Piroxicam increased glucose release and glycolysis from endogenous glycogen (glycogenolysis).
• 4.4. Gluconeogenesis from lactate plus pyruvate was inhibited.
• 5.5. The action of piroxicam on oxygen consumption was blocked by antimycin A, but not by atractyloside.
• 6.6. The action of piroxicam in the perfused rat liver metabolism seems to be a consequence of its action on mitochondria.
• 7.7. It can be concluded that inhibition of energy metabolism and stimulation of glycogenolysis are not specific properties of carboxylic nonsteroidal anti-inflammatory drugs.

     

Effect of diet and essential amino acids gavage on young rat amino acid metabolism enzymes

• 1.1. The effects of a high-fat, high-energy diet and essential plus semi-essential amino acid gavage on pup rats have been studied (60–65 animals).
• 2.2. The activities of alanine transaminase, adenylate deaminase, glutamine synthetase and serine dehydratase have been tested in liver and muscle.
• 3.3. Plasma was used for the estimation of proteins, urea, amino acids, glucose, lactate, 3-hydroxy-butyrate and acetoacetate.
• 4.4. Liver and muscle glutamine synthetase activities are increased by diet and gavage administered. Hepatic serine dehydratase is inhibited by a cafeteria diet but activated by amino acid gavage. Adenylate deaminase is inhibited by diet and gavage in the liver, but gavage does not affect this enzyme activity in muscle. Liver alanine transaminase is increased by the diet; in the muscle, cafeteria diet and amino acid gavage showed the highest values for this enzyme.
• 5.5. In the plasma, the increase in lactate produced by the diet is inhibited by the amino acids provided. Cafeteria-fed pups showed lower urea levels and higher 3-hydroxybutyrate concentrations in the plasma.
• 6.6. Intracellular glucose is diminished by cafeteria diet. In contrast, the blood cell amino acid concentration increases with diet and gavage supplied.

     

Effect of pH on metabolism of the glutamine carbon skeleton by renal cortical mitochondria

• 1.1. To determine the effect of altered acid-base homeostasis on the intramitochondrial metabolism of the glutamine carbon skeleton ¹⁴CO₂ production from [U-¹⁴C]glutamine by isolated rat renal cortical mitochondria was measured.
• 2.2. Mitochondria from rats with chronic metabolic acidosis either showed no change or diminished ¹⁴CO₂ production in comparison with pair fed controls.
• 3.3. By contrast, when the pH of the medium incubating mitochondria from normal rats was manipulated (pH 7.0, 7.4, 7.7), ¹⁴CO₂ production was clearly altered, but the direction and magnitude of the change depended on the glutamine concentration used (0.5 or 10.0 mM).
• 4.4. Mitochondria produced significant quantities of ¹⁴CO₂ when [1,4 ¹⁴C]succinate was used as substrate, indicating that ¹⁴CO₂ production from glutamine does not originate solely from the decarboxylation of α KG.
• 5.5. Thus chronic acidosis and pH, per se, affect intramitochondrial glutamine carbon skeleton metabolism in different fashions, but the specific mechanism cannot be elucidated using ¹⁴CO₂ production from [U-¹⁴C]glutamine.
• 6.6. Additional studies directly quantitating the metabolic products of glutamine have confirmed these findings and more precisely defined the sites of metabolic alteration.

     

Varying effects of calcium on the oxidation of palmitate and α-ketoglutarate in isolated rat liver mitochondria incubated in KCl-based and sucrose-based media

• 1.1. Isolated mitochondria from rat liver were incubated in the presence of [U-¹⁴C]palmitate, ATP, CoA, carnitine, EGTA (ethylene glycol bis (β-aminoethyl ether) N,N′-tetraacetic acid) and varying amounts of calcium.
• 2.2. When a KCl-based incubation medium was used, the oxidation of palmitate was inhibited when the concentration of free calcium was increased from about 0.1–10μM.
• 3.3. When a sucrose-based incubation medium was used, the basal rate of palmitate oxidation was about half of that observed with the KCl-medium and calcium had a stimulatory effect.
• 4.4. With the KCl-medium the rate of oxygen consumption was inhibited by calcium with α-ketoglutarate as well as palmitate as the respiratory substrate.
• 5.5. No inhibitory effect of calcium was observed with succinate or β-hydroxybutyrate.
• 6.6. With the KCl-medium and with α-ketoglutarate as the respiratory substrate, state 3 respiration but not state 4 respiration was inhibited by calcium.
• 7.7. When the sucrose-medium was used, state 3 respiration was first inhibited by calcium, but this inhibition was gradually relieved and the respiratory rate finally became higher than it was before calcium addition.

     

Effect of methyl methacrylate on mitochondrial function and structure

• 1.1. Treatment of isolated rat liver mitochondria with methyl methacrylate (MM) produced membrane disruption as evidenced by the release of citrate synthase, and changes in the ultrastructure of mitochondria.
• 2.2. At concentration 0.1%, MM uncoupled oxidative phosphorylation as evidenced by stimulation of state 4 respiration supported either by pyruvate plus malate or succinate (+rotenone) and ATP-ase activity in intact mitochondria.
• 3.3. At concentration 1% MM stimulated ATP-ase activity in intact mitochondria and succinate (+rotenone) oxidation at state 4 and was without effect on this substrate oxidation at state 3.
• 4.4. MM inhibited pyruvate plus malate oxidation either at state 3 or in the presence of uncoupling agents.
• 5.5. MM inhibited the NADH oxidase of electron transport particles at a concentration which failed to inhibit either succinic oxidase or the NADH-ferricyanide reductase activity.
• 6.6. The data presented suggest that in the isolated mitochondria MM inhibits NADH oxidation in the vicinity of the rotenone sensitive site of complex I.
• 7.7. The general conclusion is that MM may block an electron transport and to uncouple oxidative phosphorylation in rat liver mitochondria. The overall in vitro effect would be to prevent ATP synthesis which could result in cell death under in vivo conditions.

     

Human GMP synthetase

• 1.1. The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes. placenta, and liver.
• 2.2. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration.
• 3.3. The K_m values were determined to be 4.9μM for XMP, 270μM for ATP. and 340 μM for glutamine.
• 4.4. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient.
• 5.5. The enzyme was specific for ATP as the energy source.
• 6.6. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound.
• 7.7. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

     

Regulation of spermidine transport in l1210 cells

• 1.1. 1 mM 2-amino isobutyric add (AIB), glutamine or asparagine when preincubated for 3 hr with L1210 cells promoted a marked increase in the rate of spermidine uptake.
• 2.2. Cycloheximide also increased the transport rate and completely prevented the increase due to AIB.
• 3.3. Trifluoperazine and iso-H7 inhibited the uptake of spermidine, much less the uptake of AIB.
• 4.4. Adenosine promoted an increase in the uptake of AIB, a decrease in that of spermidine.
• 5.5. Hypotonic stress also increased the rate of spermidine transport. This modification was only partially prevented by cycloheximide.
• 6.6. Okadaic arid had no effect on this increase, whereas it prevented the increase of ODC activity.

     

Mitochondria from the ventricle of the marine clam,Mercenaria mercenaria: Substrate preferences and effects of pH and salt concentration on proline oxidation

• 1.1. Mitochondria with high respiratory control ratios (RCR) have been isolated from the ventricle of the marine clam Mercenaria mercenaria.
• 2.2. Proline is the preferred substrate of the mitochondria of the ventricle based on state 3 rates.
• 3.3. Pyruvate, ornithine and succinate are oxidized at rates 3/4 that of proline.
• 4.4. α-Glycerophosphate was oxidized at rates 1/2 that of proline.
• 5.5. The pH optimum for proline oxidation lies between 6.5 and 7.5 based on RCR and ADP/O and between 7.0 and 7.4 based on state 3 rates.
• 6.6. KCl concentrations between 250 and 450 mM gave optimal values for the oxidation of proline based on RCR and state 3 rates.
• 7.7. KCl concentration had little effect on ADP/O between 100 and 850 mM.

     

Evidence for target site insensitivity to cyanide in Spodoptera eridania larvae

• 1.1. Isolated mitochondria from the whole southern armyworm larvae, Spodoptera eridania, show all of the characteristics of mammalian liver mitochondria, except for target site sensitivity to cyanide.
• 2.2. The armyworm larval mitochondria are 17 times less sensitive to cyanide when compared to rat liver mitochondria and cannot be completely inhibited with extremely large doses.
• 3.3. These data suggest the presence in the southern armyworm of either a cyanide-insensitive cytochrome oxidase, or the elaboration of cyanide-insensitive oxidative pathway reminiscent of an alternative oxidative pathway that is known to coexist in plants alongside the cyanide-sensitive pathway.

     

Studies on lipid peroxidation in rat liver by copper deficiency

• 1.1. Copper deficiency in rats results in a 2-fold increase in the level of lipid hydroperoxides in liver mitochondria and microsomes.
• 2.2. The specific activity of cupro-zinc Superoxide dismutase decreases up to 30% while that of the mangano-enzyme is not changed.
• 3.3. Glutathione peroxidase activity as well as catalase activity are suppressed in both cytosol and mitochondrial fractions from copper-deficient rat liver.

     

Gluconeogenesis in hepatocytes determined with [2-13C] acetate and quantitative 13C NMR spectroscopy

• 1.1. In the present study the major metabolic pathways of glucose metabolism were determined in isolated liver cells using [2-¹³C]acetate and ¹³C magnetic resonance spectroscopy.
• 2.2. The relative reaction rates of glucose synthesis to the TCA cycle were determined from the ¹³C distribution in glucose where the overall ¹³C enrichment of glucose was 6.41 ± 1.94% (mean ± SD; n = 6) and the mean ¹³C enrichment of C1, C2, C5, C6 to C3, C4 was 2.63 ± 0.30.
• 3.3. Since the distribution of tracer in glucose is a function of the relative entry rates of pyruvate to acetyl-CoA into the oxaloacetate pool this was calculated to be 0.32 ± 0.15 and the factor for carbon exchange (1/P) between the gluconeogenic pathway and the TCA cycle was calculated to be 1.03 ± 0.20.
• 4.4. With this carbon exchange factor and the approximated ¹³C enrichment of acetyl-CoA the intramitochondrial ¹³C enrichment of phosphoenolpyruvate was calculated and the “true” rate of hepatic gluconeogenesis from phosphoenolpyruvate estimated.
• 5.5. Since acetate was metabolized solely in liver cells the ¹³C enrichment of acetyl-CoA could be approximated from that of 3-hydroxybutyrate.
• 6.6. The carbon 13 enrichment of 3-hydroxybutyrate and phosphoenolpyruvate was 5.89 ± 0.90% and 5.96 ± 1.67%, respectively.
• 7.7. The per cent gluconeogenesis from phosphoenolpyruvate calculated as the ratio of the ¹³C enrichment of glucose to that of 3-hydroxybutyrate times 1/P was 107 ± 8%.
• 8.8. In this study the validity of assessing isotopic exchange at oxaloacetate as suggested by Katz [Katz J. (1985) Am. J. Physiol.248, R391–R399] when interpretation of the data are not obscured by pseudoketogenesis.
• 9.9. Magnetic resonance spectroscopy provides direct information about intramolecular tracer distribution by which flux rates in major metabolic pathways are derived.

     

Role of the state of reduction of the nad system on the regulation of hepatic protein synthesis in the rat in vivo

• 1.1. The administration of octanoate to rats in vivo increased the state of reduction of the hepatic NAD system and decreased the phosphorylation potential. This effect was accompanied by a 20% inhibition of protein synthesis.
• 2.2. The acute administration of ethanol produced similar reduction of the hepatic NAD system; however, in contrast to octanoate no effect on the phosphorylation potential was detected and rates of protein synthesis were unaffected.
• 3.3. It is concluded that a rise in the state of reduction of the NAD system is not effective in decreasing hepatic protein synthesis in vivo unless it is accompanied by a decrease in the phosphorylation potential.

     

Reducing equivalent transport by substrate shuttles in rat liver and colon

• 1.1. The observed level and subcellular distribution of the α-glycerophosphate and malate-aspartate substrate shuttle enzymes in liver and colon were consistent with their proposed roles in reducing equivalent transport.
• 2.2. K_m value determinations of shuttle enzymes were performed.
• 3.3. Substrate shuttles were reconstructed from isolated liver and colon mitochondria which displayed satisfactory respiratory control and P:O ratios.
• 4.4. The results obtained suggest that while the malate-aspartate shuttle is the primary means of reducing equivalent transport in the liver, the α-glycerophosphate shuttle predominates in the colon.

     

Isolation and characterization of glutamine synthetase from the diazotroph Azospirillum brasilense

• 1.1. Glutamine synthetase was purified from the diazotroph Azospirillum brasilense.
• 2.2. The holoenzyme with a M_r of 630,000 is composed of 12 subunits of M_r 52,000.
• 3.3. A modified subunit of M_r 53,000 was also found by electrophoresis under denaturing conditions.
• 4.4. It is shown that the M_r 53,000 species is the adenylylated subunit.
• 5.5. The apparent K_m values for glutamate, ATP and ammonia were 2.5 ± 0.3 mM, 200 ± 20 μM and42 ± 2 μM, respectively.
• 6.6. Levels of glutamine synthetase activity in A. brasilense cells varied by a factor of 8 depending on the nitrogen source and its concentration in the growth medium.

     

Deleterious effects of disulfiram on the respiratory electron transport system of liver mitochondria

• 1.1. The mechanism of action of disulfiram on the respiratory electron transport system of the liver mitochondria was studied in vitro.
• 2.2. Disulfiram inhibited the respiration supported by malate-glutamate as well as succinate.
• 3.3. Mitochondrial respiration inhibition was dependent upon alteration of —SH groups.
• 4.4. The inhibitory action of disulfiram might be related to the crosslinking of several proteins of the inner mitochondrial membrane.
• 5.5. The effects described above could be attributed to disulfiram per se and not to the main metabolite diethyldithiocarbamate.

     

Effects of hyperthyroidism on glucose,glutamine and ketone-body metabolism in the gut of the rat

• 1.1. The metabolism of glucose, glutamine and ketone-bodies was studied in the small intestine of rats after 5 days of hyperthyroidism.
• 2.2. Portal-drained visceral bloodflow increased by 20.1% (P < 0.05) in hyperthyroid rats and was accompanied by a decrease in the arteriovenous concentration difference of glutamine (25.7%, P < 0.05), glutamate (22.0%, P < 0.05), alanine (20.9%, P < 0.05) and ammonia (20.6%, P < 0.05) and an increase in that of glucose (27.2%, P < 0.05), lactate (28.9%, P < 0.05) and ketone-bodies (163.2%, P< 0.001).
• 3.3. The gut of hyperthyroid rats showed increased rates of extraction of glucose, lactate and ketone-bodies.
• 4.4. Enterocytes isolated from hyperthyroid rats showed increased rates of utilization of glucose and ketone-bodies but that of glutamine were decreased.
• 5.5. The maximal activities of hexokinase, 6-phosphofructokinase, pyruvate kinase, citrate synthase and oxoglutarate dehydrogenase were increased (by 13.7–36.2%) in intestinal mucosal scrapings of hyperthyroid rats, whereas the activity of glutaminase was decreased (22.1–31.4%).
• 6.6. It is concluded that hyperthyroidism increases the rates of utilization of glucose and ketone-bodies but decreases that of glutamine (both in vivo and in vitro) by the epithelial cells of the small intestine.

     

Intramitochondrial reductive carboxylation of 2-oxoglutarate in adipose tissue and its contribution to fatty acid synthesis

• 1.1. The reductive carboxylation of 2-oxoglutarate was found to proceed in mitochondria of rat epididymal fat pads and rabbit perirenal adipose tissue at a rate similar to that in liver mitochondria.
• 2.2. In rat fat pads the incorporation of ¹⁴C from [5-¹⁴C]2-oxoglutarate into fatty acids via the carboxylation was suppressed by butylmalonate by 30%.
• 3.3. 2-Oxoglutarate and glutamate stimulated the incorporation into fatty acids of ¹⁴C from [2-¹⁴C]acetate in rat fat pads with the simultaneous reduction of tissue NADP. These effects persisted after inhibition of succinate dehydrogenase by malonate.
• 4.4. It is concluded that in adipose tissue 2-oxoglutarate carboxylation proceeds in both the cytoplasm and mitochondria. Therefore, it can supply carbon atoms as well as NADPH for fatty acid synthesis.