Sodium benzoate inhibits fatty acid oxidation in rat liver: effect on ammonia levels

Sodium benzoate inhibited PC and octanoic acid-mediated State 3 respiration rates by 39 and 29%, respectively, at 0.5 mM in isolated rat liver mitochondria. At 2 mM, benzoate did not affect State 3 respiration rates with either succinate or malate plus glutamate, indicating that it did not act as an uncoupler. The oxidation of palmitate and octanoate was inhibited by 39 and 54% at 2 mM benzoate in liver homogenates. Benzoate, at 10 mmol/kg caused significant decreases in the levels of hepatic ATP, CoA, and acetyl-CoA. Administration of sodium benzoate to rats caused a dose-dependent increase in hepatic ammonia levels. However, the inhibitory effect of benzoate on fatty acid oxidation is not mediated through ammonia since ammonium chloride, at 1 mM, did not inhibit PC or octanoate oxidation in mitochondria or their oxidation in liver homogenate. Our results warrant a reevaluation of the use of sodium benzoate in the treatment of hyperammonemia.     

Pathway of glutamate oxidation and its regulation in the HuH13 line of human hepatoma cells.

Well-coupled mitochondria were isolated from a HuH13 line of human hepatoma cells and human liver tissue. The liver mitochondria showed a feeble glutamine oxidation activity in contrast to the hepatoma mitochondria, whereas they utilized glutamate well for the oxidative phosphorylation. In the liver mitochondria, glutamate was oxidized via the routes of transamination and deamination. On the other hand, glutamate oxidation was initiated preferentially via transamination pathway in the tumor mitochondria. In the liver mitochondria, bicarbonate nearly at a physiological concentration inhibited oxygen uptake with glutamate as substrate. The interaction of bicarbonate with the pathway of glutamate oxidation occurred primarily at the level of succinate dehydrogenase, due to competitive inhibition of the enzyme by the compound. In contrast to the liver mitochondria, glutamate oxidation was not affected by bicarbonate in the tumor mitochondria. These results indicate that the aberrations in the glutamate metabolism and its regulation observed in the hepatoma mitochondria may be favorable to the respiration utilizing glutamine and/or glutamate as an energy source.     

Effects of peginol C-2000 on respiration and oxidative phosphorylation in liver mitochondria of rats poisoned with butylcaptax]

The data on the effect of synthetic antioxidant Paginol C-2000 on respiration and oxidative phosphorylation in liver mitochondria of rats primed with butylcaptax are given. It is shown that intoxication of organism with butylcaptax in the dose of 1/10 LD50 during 5 days resulted in suppression of respiration and oxidative phosphorylation in rat liver mitochondria. It is found, that administration of Raginol C-2000 into organisms of primed animals leads to the reduction of succinate pathway of oxidation, that, in its turn, results in the normalization of functioning level of ATP-synthesizing system of mitochondria.     

Activation succinate dehydrogenation in rat liver by noradrenaline, cAMP and acute cooling

Administration of noradrenaline in a dose of 11 mumol/kg activates succinate dehydrogenase (SDH) in the rat liver. On incubation of liver homogenate with noradrenaline (10(-6) M), cAMP (10(-6) M) and NaF (10(-2) M), a nonhormonal activator of adenylate cyclase, SDH was found to be activated by 20--30% in all the cases. The effects of noradrenaline and cAMP did not sum up. It seems likely that SDH activation by catecholamines is mediated by cAMP. Acute cooling (--17 degrees C) stimulates SDH via endogenous catecholamines, since this effect is completely reversed by the beta-adrenolytic inderal.     

Polarographic observation of substrate-level phosphorylation and its stimulation by acetylcholine

Substrate-level phosphorylation was observed under the conditions optimal for this process and opposite to those for oxidative phosphorylation. Polarographic registration of Ca2+ stimulated alpha-ketoglutarate oxidation and self-inhibition of uncoupled alpha-ketoglutarate (KG) oxidation was used. Acetylcholine (ACh) administration stimulated KG oxidation and substrate-level phosphorylation in isolated mitochondria. These effects are stronger in tissues with a higher level of endogenous acetylcholine, such as guinea pig liver vs rat liver and pancreas vs liver. The specific stimulation of KG oxidation by ACh is related to a decrease of succinate oxidation and is contrary to the specific stimulating effect of adrenaline on succinate oxidation. Therefore the existence of reciprocal hormone-substrate-nucleotide systems is suggested. The described set of conditions optimal for substrate-level phosphorylation observation by polarographic registration of respiration is as convenient as the ADP test for the investigation of oxidative phosphorylation.     

Preservation of native properties of mitochondria in rat liver homogenate

A protocol is developed for preparation of concentrated rat liver homogenate preserving assemblies of mitochondria in isotonic KCl under 0 and 15 degrees C. Assemblies preserve ability for self-organization during storage in homogenate. All key energy functions of mitochondria can be investigated in such a homogenate. Oxidative phosphorylation and membrane potential are stable for 5-7 h and can be still observed on the next day. Substrate-level phosphorylation is better pronounced for mitochondria in KCl than in sucrose medium while Ca2+ capacity is greater and lipid peroxidation is much lower. Sucrose addition impairs these functions. The rate of phosphorylating respiration is lower in large assemblies and higher in small. Transition from large to small assemblies corresponds to the transition from quiescent state of animal to adrenaline induced active state. The proposed method is particularly convenient for clinical investigations with small bioptates.     

Mitochondrial respiration and triiodothyronine concentration in liver from postpubertal and adult rats.

The purpose of this study was to investigate the decline in rat liver mitochondria respiration found in adult rats compared to younger ones, and to find a link between this respiratory impairment and a tissue hypothyroidism state. To this end, hepatic concentration and serum levels of triiodothyronine were measured in postpubertal rats (60 days old) and adult rats (180 days old). In addition, in these rats we measured oxidative phosphorylation in homogenate together with coupled and uncoupled respiration in isolated mitochondria using succinate or durohydroquinone as substrate. We found that mitochondria from adult rats consumed less oxygen compared to younger rats due to lower electron transport chain and phosphorylating system activity. In addition, we found that in state 4 condition, mitochondria from adult rats consumed less oxygen than mitochondria from young rats. Finally, we found a decrease in liver triiodothyronine concentration in adult rats. In conclusion, the results of this study show that hepatic mitochondria in adult rats have a decreased ATP synthesis capacity and proton permeability, both consistent with the tissue hypothyroidism found in the liver of adult rats.     

Amaranth seed oil: Effect of oral administration on energetic functions of rat liver mitochondria activated with adrenaline

Respiration parameters of liver mitochondria (MCh) in rats fed with amaranth seed oil for 3 weeks have been evaluated. Thirty minutes before decapitation, adrenaline was injected intraperitoneally at a low dose (350 μg/kg body weight) to both control and experimental animals. It was shown that in animals that were injected with adrenaline and did not receive oil, the rate of phosphorylating respiration increased by 32% and phosphorylation time decreased by 22% upon oxidation of succinate; upon oxidation of α-ketoglutarate in the presence of the succinate dehydrogenase inhibitor malonate, phosphorylating respiration was activated by 23%. The respiration of MCh upon oxidation of succinate + glutamate and α-ketoglutarate in the absence of malonate was not affected by adrenaline. The intake of oil markedly activated almost all parameters of mitochondrial respiration in experimental rats upon oxidation of all above-listed substrates in both coupled and uncoupled MCh. However, phosphorylation time was close to the control value (upon oxidation of succinate) or increased (upon oxidation of α-ketoglutarate in the presence and absence of malonate). The injection of adrenaline to animals receiving oil did not affect the oil-activated respiration of MCh oxidizing the substrates used; however, phosphorylation time in all groups of animals decreased. Ca²⁺ capacity of MCh in rats receiving amaranth oil did not change. Thus, our data show that feeding of rats with amaranth oil activates mitochondrial respiration and prevents MCh hyperactivation induced by adrenaline.     

The effect of low concentrations of sodium nitrites and nitrates on respiration and oxidative phosphorylation in rat liver mitochondria]

Nitrite incubation in mitochondria and nitrate intoxication of rats have been studied for their effect on aerobic energetic processes in the liver. Sodium nitrite in concentration of 2 mg/l causes an inhibition of ADP-stimulated respiration and provides uncoupling processes of oxidative phosphorylation and respiration in mitochondria, when adding succinate as a substrate. Low doses of nitrate in vivo promote oxygen economization in mitochondria. Intoxication of rats with nitrate in a dose of 50 mg/l for 30 days induces a decrease of the respiration rate after ADP-phosphorylation and an increase of the coefficient of oxidative phosphorylation efficiency (ADP/O). Intraperitoneal administration of adrenalin in a dose of 25 micrograms/100 g to rats after 30-day nitrate intoxication in a concentration of 10 mg/l induces no typical increase of ADP-stimulated respiration and rate of oxidative phosphorylation and succinate oxidation.     

Tissue-specific regulation of rat liver mitochondrial oxidative phosphorylation by a soluble phase of cells from that organ

Tissue specificity of mitochondrial respiration stimulation under the effect of a soluble phase of liver cells (SPC) is preserved by addition to dinitrophenol but is reserved in the presence of oligomycin. Addition of rotenon in the presence of SPC entails a tissue-specific increase in respiration that is proportional to the respective increase in respiration of intact mitochondria in the presence of the inhibitor mentioned. SPC tissue-specifically inhibits ATPase activity of liver mitochondria. This fraction of SPC is capable of recovering the coupling of oxidative phosphorylation of mitochondria whose respiration is inhibited by adding ADP. A conclusion is made that SPC is capable not only to decrease tissue-specifically the coupling of intact mitochondria but also to raise it in mitochondria with deranged oxidative phosphorylation. This assures intratissue organization of liver metabolism by means of tissue-specific stabilization of liver cell energy metabolism.     

Oxidation of lactate in isolated rat heart mitochondria

In unwashed mitochondria the oxidation of L-lactate (with NAD+) proceeds in presence of the added lactate dehydrogenase. The respiration is characterized by the high rate in state 4 and is stimulated by ADP. This process takes place in unwashed mitochondria and homogenate of the heart in absence of added lactate dehydrogenase. Oxidation of lactate with NAD+ is inhibited by rotenone. It has been also revealed that the oxidation of glutamate is insufficiently altered in presence of lactate (with NAD+) in unwashed mitochondria as compared with the washed ones. It is supposed that the stimulating effect of lactate with NAD+ on the mitochondria respiration is not so much a result of the membrane-damaged action as a result of oxidation of lactate dehydrogenase reaction products: phosphorylative oxidation of pyruvate and nonconjugated oxidation of NADH. Utilization of these products takes place in the main respiratory chain, including its first stage.     

The effect of metabolic inhibitors on the development of respiration in anaerobically grown yeast

1. Iodoacetate and fluoride did not prevent the development of respiration in aerobically grown yeast. 2. The effect of dinitrophenol suggested that phosphorylation developed simultaneously with respiration in anaerobically grown yeast, but the effect of oligomycin suggested that the phosphorylation and oxidation were not tightly coupled. 3. Inhibitors of electron transport showed that both the respiratory peak and the subsequent respiration were cyanide-sensitive, but the peak respiration was insensitive to antimycin. 4. Of the inhibitors of protein or RNA synthesis tested, only p-fluorophenylalanine inhibited the development of respiration. The results are not consistent with a new synthesis of mitochondria. 5. 2-Phenylethanol inhibited the development of respiration in anaerobically grown yeast and also yeast growth. Other inhibitors of DNA synthesis had no effect on the development of respiration. 6. The relevance of the results to mitochondrial morphogenesis is discussed.     

The relationship between plasma free fatty acids and liver mitochondrial function in vivo

P/O ratio, state 3 and 4 respiration rates, and acceptor control index (ACI) were assessed in rat liver mitochondria following an overnight fast and single bout of treadmill exercise of 30-180 min. P/O was unaffected by fasting and 30 min of exercise; however, ACI was reduced because of an increase in state 4 respiration. Fasting, followed by running for 1 h or more decreased P/O approx. 40% and ACI by 50%, an effect that could be attributed to a reduction in state 3 respiration. The decrease in P/O was reversed 15 min after the cessation of exercise, whereas ACI remained depressed. All these functional alterations were mimicked by incubation of isolated mitochondria with palmitate and reversed by washing them with albumin. No direct correlation between plasma free fatty acids and the alterations in mitochondrial respiration was apparent. These data demonstrate that the decrease in the normal coupling of oxidation and phosphorylation in liver mitochondria produced by fasting/exercise is reversed rapidly in vivo. Furthermore, it is apparent that, if fatty acids act as a regulatory agent under these conditions, they do not do so solely on the basis of their plasma concentration.     

The effect of noradrenaline on glyceride synthesis and oxidative metabolism in vitro in the brown fat of newborn rabbits

1. The effect of noradrenaline on the synthesis of glyceride from [U-¹⁴C]glucose and on gas exchange in the brown fat of newborn rabbits in vitro was investigated. 2. The specific radioactivity of l-glycerol 3-phosphate was lower than that of lactate, presumably because glycerol derived from glyceride was rephosphorylated by glycerokinase. 3. In the basal state more than 25% of the total respiration was due to pyruvate oxidation. Noradrenaline stimulated glyceride synthesis and total respiration without changing the proportion of the total respiration due to pyruvate oxidation. 4. The extra ADP released by noradrenaline stimulation of glyceride synthesis could not have supported more than 2% of the observed increase in substrate oxidation if mitochondria from brown-fat-cells remain fully coupled in the stimulated state, but could have supported about one-third of the observed increase if they become uncoupled in the presence of noradrenaline.     

Activation of respiration in liver mitochondria by catecholamines]

Catacholamines activate in vivo and in vitro the rat liver mitochondria respiration (VO2) at high piruvate, 2-oxoglutarate and succinate concentrations. The effect was equally characteristic of free oxidation (state 4), phosphorylation (state 3), and even of VO2 uncoupled by DNP. EDTA and bovine serum albumin addition failed to eliminate the effect of the oxidoreductases activation by catecholamines.     

The acute regulation of mitochondrial proton conductance in cells and mitochondria from the brown fat of cold-adapted and warm-adapted guinea pigs

Cells and mitochondria were prepared from the brown adipose tissue of adult guinea-pigs adapted to either 4-7 degrees C or 22-25 degrees C. The cold-adapted cells displayed noradrenaline-stimulated, propranolol-sensitive respiration, but noradrenaline failed to increase the respiration of the warm-adapted cells. Purine-nucleotide-sensitive proton conductance was greater in cold-adapted mitochondria than in warm-adapted controls. At the same time cold-adapted mitochondria were extremely sensitive to the uncoupling effect of endogenous and infused fatty acids, and resembled the mitochondria from the brown adipose tissue of cold-adapted hamsters. Warm-adapted mitochondria were ninefold less sensitive, and resembled liver mitochondria. With cold-adapted, but not warm-adapted mitochondria, respiration increased proportionately to the rate of fatty acid infusion. It is concluded that the presence of the 32000-Mr proton conductance pathway is necessary for the expression of a high sensitivity to fatty acid uncoupling, suggesting that the fatty acids interact directly with this protein to modulate the proton conductance during the acute regulation of thermogenesis.     

Effect of protein-markers of growth on oxidative phosphorylation in mitochondria

Growth marker proteins (GMP) were studied for their effect on oxidative phosphorylation in the heart and liver mitochondria of rabbits. It is shown that GMP decrease a respiratory control (RC) coefficient, P/O coefficient, inhibit respiration of the mitochondria in metabolic states 3, 5 and activates it in state 4. The nature of the oxidation substrates (FAD- and NAD-dependent succinic and pyruvic acids, respectively) does not influence the GMP effect manifestation. It is supposed that GMP disturb the structural and functional integrity of the mitochondria. Variations in bioenergetic parameters of the heart and liver mitochondria in organisms with active growth foci as well as of mitochondria incubated with GMP, are unidirectional. Cytochrome c, coenzyme A (Co ASH) and other thyol compounds (cystein, dithiotreitol, glutathione--GSH) remove the GMP action.     

The effect of hypoxia on oxidative phosphorylation and lipid peroxidation in rat liver mitochondria upon lung inflammation

Hypoxic trainings of rats (maintenance in the test chamber at the "altitude" 4 km above the sea level for 7 hours a day for two weeks) prevent pneumonia-induced activation of peroxidation for lipids of the liver mitochondria. This increases the phosphorylating respiration rate when lipemic serum is used as an oxidation substrate (not succinate). In these experiments the efficiency of oxidative phosphorylation (delta ADP/delta O) when either succinate and glutamate or glutamate and malate have been oxidated corresponded to the values typical of intact animals and was higher when lipemic serum was used. It is supposed that rearrangement of energy reactions of mitochondria is connected with intensification of utilization of lipids and conjugation between their oxidation and phosphorylation. This rearrangement is apparently aimed to prevent the energy deficiency in the organism which arises in patients with pneumonia.     

Mitochondrial metabolic suppression and reactive oxygen species production in liver and skeletal muscle of hibernating thirteen-lined ground squirrels

During hibernation, animals cycle between periods of torpor, during which body temperature (T(b)) and metabolic rate (MR) are suppressed for days, and interbout euthermia (IBE), during which T(b) and MR return to resting levels for several hours. In this study, we measured respiration rates, membrane potentials, and reactive oxygen species (ROS) production of liver and skeletal muscle mitochondria isolated from ground squirrels (Ictidomys tridecemlineatus) during torpor and IBE to determine how mitochondrial metabolism is suppressed during torpor and how this suppression affects oxidative stress. In liver and skeletal muscle, state 3 respiration measured at 37°C with succinate was 70% and 30% lower, respectively, during torpor. In liver, this suppression was achieved largely via inhibition of substrate oxidation, likely at succinate dehydrogenase. In both tissues, respiration by torpid mitochondria further declined up to 88% when mitochondria were cooled to 10°C, close to torpid T(b). In liver, this passive thermal effect on respiration rate reflected reduced activity of all components of oxidative phosphorylation (substrate oxidation, phosphorylation, and proton leak). With glutamate + malate and succinate, mitochondrial free radical leak (FRL; proportion of electrons leading to ROS production) was higher in torpor than IBE, but only in liver. With succinate, higher FRL likely resulted from increased reduction state of complex III during torpor. With glutamate + malate, higher FRL resulted from active suppression of complex I ROS production during IBE, which may limit ROS production during arousal. In both tissues, ROS production and FRL declined with temperature, suggesting ROS production is also reduced during torpor by passive thermal effects.     

Evidence of a phosphate-transporter system in the inner membrane of isolated mitochondria

1. The organic mercurial sodium mersalyl, formaldehyde, dicyclohexylcarbodiimide and tributyltin each blocked respiratory-chain-linked ATP synthesis in rat liver mitochondria. 2. Mersalyl and formaldehyde also blocked a number of other processes dependent on the entry of inorganic phosphate into mitochondria, including mitochondrial respiration and swelling stimulated by cations and phosphate, the substrate-level phosphorylation reaction of the citric acid cycle, and swelling in ammonium phosphate. 3. Dicyclohexylcarbodi-imide and tributyltin did not inhibit the entry of phosphate into mitochondria. 4. Mersalyl and formaldehyde had a relatively slight effect on succinate oxidation and swelling stimulated by cations when phosphate was replaced by acetate, on succinate oxidation stimulated by uncoupling agents, and on swelling in solutions of ammonium salts other than phosphate or arsenate. 5. Formaldehyde blocked the oxidation of NAD-linked substrates in mitochondria treated with 2,4-dinitrophenol and the ATP-dependent reduction of NAD by succinate catalysed by ox heart submitochondrial particles. Both these effects appear to be due to an inhibition by formaldehyde of the NAD-flavin region of the respiratory chain. 6. Concentrations of dicyclohexylcarbodiimide or tributyltin sufficient to abolish ADP-stimulated respiration blocked the dinitrophenol-stimulated adenosine triphosphatase activity, whereas mersalyl and formaldehyde caused only partial inhibition of ATP hydrolysis. 7. When mitochondria were incubated with dinitrophenol and ATP, less than 10% of the total inorganic phosphate liberated was recovered in the mitochondria and no swelling occurred. In the presence of mersalyl or formaldehyde at least 80% of the total inorganic phosphate liberated was retained in the mitochondria and extensive swelling was observed. This swelling was inhibited by oligomycin but not by antimycin or rotenone. 8. The addition of mersalyl to mitochondria swollen by treatment with valinomycin, K(+) and phosphate blocked the contraction induced by dinitrophenol and caused an increase in the phosphate content of the mitochondria, but had no effect on the contraction of mitochondria when phosphate was replaced by acetate. 9. It is concluded that mitochondria contain a phosphate-transporter system, which catalyses the movement of phosphate in either direction across the mitochondrial membrane, and that this system is inactivated by organic mercurials and by formaldehyde. Evidence is presented that the phosphate-transporter system is situated in the inner membrane of rat liver mitochondria and is also present in other types of mammalian mitochondria.