Metabolic state of liver mitochondria and lymphocytes after T-activin administration under condition of hypothermia

After 10 days of swimming (10 min per day, water temperature +20 degrees C) the oxygen consumption in rat liver mitochondria increased via the external pathway of NADH oxidation from 3.6 +/- 0.3 to 4.4 +/- 0,2 nmoles O2 x min-1 x mg-1 protein; when the rats were simultaneously injected with an endogenous immunomodulator T-activin (5 micrograms/100 g body weight) daily, this rate increased up to 6.5 +/- 0.6 nmoles O2 x min-1 x mg-1 protein. In the control group, the uncoupled respiration rate is also higher, while the ascorbate+ +TMPD oxidation rate is lower than in the cold- and cold + T-activin-treated groups. The metabolic states of lymphocyte mitochondria did not differ in the three experimental groups. The respiration rates and delta psi m (monitored by diS-C3-(5) fluorescence) of lymphocyte mitochondria in these three groups were also identical.     

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.     

Role of the Ca2+ cycle in uncoupling of oxidative phosphorylation in liver mitochondria of cold-acclimated rats

Cold acclimation of Wistar rats for 2-4 weeks at about 3 degrees C resulted in an increased respiration rate and a reduced ADP/O ratio in liver mitochondria. With increasing duration of acclimation up to 10-12 weeks, these parameters returned to a normal level. The increase in the respiration rate and the decline of the mitochondrial ADP/O ratio were associated with a significant activation of the electroneutral release of Ca2+. When the animals were acclimated for 10-12 weeks the rate of Ca2+ release reduced to control values. The addition of 1 microM ruthenium red resulted in a decrease in the rates of mitochondrial respiration in control and cold-acclimated rats to approximately equal values and in a partial restoration of the ADP/O ratio in liver mitochondria of rats kept in the cold for 2-4 weeks. The respiratory activity of mitochondria isolated in the presence of 1 mM EGTA unaffected by ruthenium red.     

Corrections by melatonin of liver mitochondrial disorders under diabetes and acute intoxication in rats

The aim of the present work was to investigate the mechanisms of oxidative damage of the liver mitochondria under diabetes and intoxication in rats as well as to evaluate the possibility of corrections of mitochondrial disorders by pharmacological doses of melatonin. The experimental (30 days) streptozotocin‐induced diabetes mellitus caused a significant damage of the respiratory activity in rat liver mitochondria. In the case of succinate as a respiratory substrate, the ADP‐stimulated respiration rate V₃ considerably decreased (by 25%, p < 0·05) as well as the acceptor control ratio (ACR) V₃/V₂ markedly diminished (by 25%, p < 0·01). We observed a decrease of the ADP‐stimulated respiration rate V₃ by 35% (p < 0·05), with glutamate as substrate. In this case, ACR also decreased (by 20%, p < 0·05). Surprisingly, the phosphorylation coefficient ADP/O did not change under diabetic liver damage. Acute rat carbon tetrachloride‐induced intoxication resulted in considerable decrease of the phosphorylation coefficient because of uncoupling of the oxidation and phosphorylation processes in the liver mitochondria. The melatonin administration during diabetes (10 mg·kg^‐1 body weight, 30 days, daily) showed a considerable protective effect on the liver mitochondrial function, reversing the decreased respiration rate V₃ and the diminished ACR to the control values both for succinate‐dependent respiration and for glutamate‐dependent respiration. The melatonin administration to intoxicated animals (10 mg·kg⁻¹ body weight, three times) partially increased the rate of succinate‐dependent respiration coupled with phosphorylation. The impairment of mitochondrial respiratory plays a key role in the development of liver injury under diabetes and intoxication. Melatonin might be considered as an effector that regulates the mitochondrial function under diabetes. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of ethanol intake on rat liver mitochondrial respiration and oxidative phosphorylation

The effect of ethanol intake on liver mitochondrial functions was investigated by feeding rats with a liquid isocaloric diet containing various concentrations of ethanol. We found that after feeding the liquid diet for 2 to 3 months, the body weight of rats did not show a significant difference between treated and control groups. However, the mitochondrial respiration rate decreased significantly with the increase of ethanol concentration in the diet. We found that when the rats were fed on 10.8% ethanol, the average succinate-supported State 3 respiration rate decreased from 54.5 to 44.8 nmol O2/min/mg and the glutamate-malate-supported State 3 respiration rate decreased from 38.8 to 23.6 nmol O2/min/mg as compared with the control. Interestingly, we noted that ethanol intake caused a more drastic effect on State 3 respiration than on State 4 respiration, irrespective of the substrate utilized by the mitochondria. In addition, the respiratory control and ADP/O ratios were found to decrease concomitantly with the increase of ethanol level in the diet. Moreover, we found that the effect of ethanol on both respiratory control and ADP/O ratios of liver mitochondria was more pronounced in glutamate-malate-supported respiration than succinate-supported respiration. These results clearly demonstrate that ethanol intake by the rat can cause impairment of liver mitochondrial respiration and oxidative phosphorylation, and that these effects are exerted through damage to mitochondrial membranes.     

The oxidation of different substrates by liver mitochondria from rat pups in early postnatal development

We have studied the respiratory ratio of liver mitochondria during oxidation of substrates of the carbohydrate and lipid metabolism in newborn rats and during early postnatal development. High rate of respiration coupled with the synthesis of ATP from ADP and phosphate has been found during oxidation of carbohydrate substrates (pyruvate + malate); however, caprilate, a substrate of lipid metabolism, does not support such respiration. However, in the young rats aging from 2 to 30 days utilization of carbohydrate and lipid substrates via the phosphorylating pathway proceeds with similar efficacy.     

Stimulation of the respiratory and phosphorylating activities in rat brain mitochondria by idebenone (CV-2619), a new agent improving cerebral metabolism

The effects of 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (CV-2619) on the respiration and non-respiratory oxygen consumption induced by ascorbate and Fe2+ in rat brain mitochondria were studied. When CV-2619 (100 and 300 mg/kg) was orally administered to rats for 3 days, it increased the state 3 respiration stimulated by ADP, slightly decreased the state 4 respiration after the consumption of ADP and resulted in a significant increase of the respiratory control index (RCI) by 14-19% for glutamate oxidation (p less than 0.01) and 10-17% for succinate oxidation (p less than 0.05), respectively. The RCI increasing effect of CV-2619 was dose dependent, but the compound had no effect on the ADP/O ratio. CV-2619 significantly suppressed by about 10% the non-respiratory oxygen consumption (p less than 0.02), which closely associated with non-enzymatic reactions such as lipid peroxidation, membrane lysis and swelling of mitochondria. Thus, CV-2619 may contribute to stimulate the net ATP formation by the well-coupling of electron and energy transfer, and by the reduction of non-respiratory oxygen consumption in cerebral metabolism.     

Food restriction affects energy metabolism in rat liver mitochondria

To examine the effect of 50% food restriction over a period of 3 days on mitochondrial energy metabolism, liver mitochondria were isolated from ad libitum and food-restricted rats. Mitochondrial enzyme activities and oxygen consumption were assessed spectrophotometrically and polarographically. With regard to body weight loss (-5%), food restriction decreased the liver to body mass ratio by 7%. Moreover, in food-restricted rats, liver mitochondria displayed diminished state 3 (-30%), state 4-oligomycin (-26%) and uncoupled state (-24%) respiration rates in the presence of succinate. Furthermore, "top-down" elasticity showed that these decreases were due to an inactivation of reactions involved in substrate oxidation. Therefore, it appears that rats not only adapt to food restriction through simple passive mechanisms, such as liver mass loss, but also through decreased mitochondrial energetic metabolism.     

Effect of exhaustive exercise on liver mitochondrial function in the rat

The oxidative and phosphorylative function of rat liver mitochondria after exhaustive exercise was investigated. The stimulation of state 4 respiration (without ADP) with NADH and FADH2 dependent substrates was demonstrated. The reduction in RCR ratio (the rate of oxidation in state 3/the rate of oxidation in state 4) and enhanced activity of oligomycin sensitive ATP-ase was also found. The results suggest an inhibition of liver mitochondrial phosphorylative activity in rats exercised till exhaustion.     

The possible role of palmitoyl-CoA in the regulation of the adenine nucleotides transport in mitochondria under different metabolic states. I. Comparison of liver mitochondria from starved and fed rats.

It has been shown that KM values for ADP when rat liver mitochondria oxidized succinate were strictly dependent on the values of the respiratory control ratios. The Ki values for palmitoyl-CoA inhibition of the ADP-stimulated succinate oxidation and the inhibition of the uncoupler-stimulated ATPase activity were equal to 0.5 muM. Mitochondria from livers of starved rats showed 30% inhibition of the state 3 respiratory rate (compared to the uncoupled respiratory rate) which was abolished by addition of carnitine. It was supposed that this inhibition was due to the influence of acyl-CoAs bound to the inner mitochondrial membrane on the adeninenucleotide translocase. Mitochondria from livers of fed rats showed a strong inhibition of succinate oxidation both in state 4 and state 3, although the rate of uncoupled respiration was normal. It was assumed that in this case the changes in mitochondrial behaviour was caused by the decrease in the concentration of ADP and ATP in the matrix space of mitochondria.     

Effect of water and food deprivation in pregnant rats on mitochondrial respiration and the state of gluconeogenesis in the rat pup liver

Wistar female rats were submitted to a water and food deprivation (food and water were restricted by 50% as compared to those consumed by control animals) during the entire pregnancy. There were 6 to 8 pups in the litters of experimental dams, the body weight of these pups being significantly lower than in control. In liver homogenates from deprived animals, intensity of gluconeogenesis was decreased by 50% or more, and gluconeogenesis precursors did not stimulate the glucose synthesis. Oxidation of pyruvate and malate as well as of caprylate by liver mitochondria remained significantly lower than in control from the postnatal day 10 to 2 months. A possible role of energetic disturbances in underdevelopment of animals submitted to the water and food deprivation during the intrauterine development is considered.     

Effect of water and food deprivation in pregnant rats on mitochondrial respiration and the state of gluconeogenesis in the rat pup liver

Wistar female rats were submitted to a water and food deprivation (food and water were restricted by 50% as compared to those consumed by control animals) during the entire pregnancy. There were 6 to 8 pups in the litters of experimental dams, the body weight of these pups being significantly lower than in control. In liver homogenates from deprived animals, intensity of gluconeogenesis was decreased by 50% or more, and gluconeogenesis precursors did not stimulate the glucose synthesis. Oxidation of pyruvate and malate as well as of caprylate by liver mitochondria remained significantly lower than in control from the postnatal day 10 to 2 months. A possible role of energetic disturbances in underdevelopment of animals submitted to the water and food deprivation during the intrauterine development is considered.     

Control of adenine nucleotide metabolism in hepatic mitochondria from rats with ethanol-induced fatty liver

Male rats developed fatty liver after being fed on an ethanol-containing diet for 31 days. Liver mitochondria from these animals catalysed ATP synthesis at a slower rate when compared with mitochondria from pair-fed control rats (control mitochondria), and demonstrated lowered respiratory control with succinate as substrate, owing to a decrease in the State-3 respiratory rate. Respiration in the presence of uncoupler was comparable in mitochondria from both groups of rats. Translocation of both ATP and ADP was decreased in mitochondria from ethanol-fed rats, with ADP uptake being lowered more dramatically by ethanol feeding. Parameters influencing adenine nucleotide translocation were investigated in mitochondria from ethanol-fed rats. Experiments performed suggested that lowered adenine nucleotide translocation in these mitochondria is not the result of inhibition of the translocase by either long-chain acyl-CoA derivatives or unesterified fatty acids. Analysis of endogenous adenine nucleotides in these mitochondria revealed lowered ATP concentrations, but no decrease in total adenine nucleotides. In experiments where the endogenous ATP in these mitochondria was shifted to higher concentrations by incubation with oxidizable substrates or defatted bovine serum albumin, the rate of ADP translocation was increased, with a linear correlation being observed between endogenous ATP concentrations and the rate of ADP translocation. The depressed ATP concentration in mitochondria from ethanol-fed rats suggests that the ATP synthetase complex is replenishing endogenous ATP at a slower rate. The lowered ATPase activity of the ATP synthetase observed in submitochondrial particles from ethanol-fed animals suggests a decrease in the function of the synthetase complex. A decrease in the rate of ATP synthesis in mitochondria from ethanol-fed rats is sufficient to explain the decreased ADP translocation and State-3 respiration.     

Progesterone mediated increase in monoamine stores and the regulation of enzymes of biosynthesis and metabolism in the adrenal gland during late pregnancy in the rat.

The influence of repeated injections of progesterone to pregnant rats upon monoamine storage and regulation of enzymes phenylethanolamine-N-methyltransferase (PNMT), monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) was studied. All the pregnant females received progesterone (4 mg/100 g body weight) on 19, 20 and 21 days post-coitum but one group was killed at 21 days of pregnancy and the other one at 0 h parturition. Adrenal epinephrine demonstrated highly significant increase in progesterone treated rats. At the same time norepinephrine content declined significantly from the control value. The activity of enzyme PNMT also showed marked increase in the adrenals of progesterone treated females. Activity of enzyme MAO showed a slight decline after progesterone treatment to pregnant rats. Enzyme COMT in progesterone treateed animals showed decline at 0 h parturition but at 21 days post-coitum it was significantly higher from non-injected females. All the increases and decreases in monoamines and the three enzymes were significant when the results were expressed per adrenal gland or per gram of adrenal. The results suggest that exogenous progesterone administration during late pregnancy increases epinephrine stores by declining monoamine metabolism by MAO and COMT and increasing their synthesis by PNMT which is responsible for N-methylation of norepinephrine to epinephrine.     

The characteristics of lactate oxidation in the tissue mitochondria of rats of different ages]

The parameters of respiration (V3, V4) and phosphorylation (the respiration control, ADP/O) have been studied using lactate as a substrate (obligatory with NAD addition) close by meaning to pyruvate on the liver and heart mitochondrion and homogenates of newborn rats. In 20-days and adult rats the mitochondria and homogenates oxidize the lactate (with NAD) with higher rate V4 but with lower value of respiration control as compared with the newborn animals. Simultaneously, a high activity of mitochondrial NADH-oxidase, oxidizing NADH, formed in the reaction of lactate dehydrogenase not connected with ATP synthesis. The role of mitochondrial NADH-oxidase are discussed as a factor increasing lactate oxidation, removing tissue lactate and activating the age dependent energy metabolism.     

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.     

Oxidative activity in mitochondria isolated from rat liver at different stages of development

The purpose of this study was to evaluate the oxidative capacities in hepatic mitochondria isolated from prepubertal, young adult and adult rats (40, 90 and 180 days of age, respectively). In these rats, mitochondrial respiratory rates using FAD- and NAD-linked substrates as well as mitochondrial protein mass were measured. The results show that only the oxidative capacity of FAD-linked pathways significantly declined in mitochondria from 180-day-old rats compared with those from younger animals. When we consider FAD-linked respiration expressed per g liver, no significant difference was found among rats of different ages because of an increased mitochondrial protein mass found in 180-day-old rats. However, when FAD-linked and lipid-dependent respiratory rates were expressed per 100 g body weight, significant decreases occurred in 180-day-old rats. Therefore, the decrease in liver weight expressed per 100 g body weight rather than an impaired hepatic cellular activity may be the cause of body energy deficit in 180-day-old rats. Copyright © 1998 John Wiley & Sons, Ltd.     

Acute and chronic hypoxia in rats. II. Effect on oxidative phosphorylation and in vitro protein synthesis in liver mitochondria and its subpopulations.

The objective of this investigation was to examine liver mitochondrial functions in rats exposed to 0.4 atm for 0, 5 and 27 days, Liver homogenates were fractionated by rate-zonal centrifugation utilizing iso-osmotic Ficoll-sucrose gradients; this eliminates loss of large and small mitochondria and makes possible the separation of mitochondria into subpopulations according to sedimentation coefficient. After pooling all mitochondrial fractions for obtaining composite determinations of the entire population, large diminutions in states 3 and 4 respiration (succinate as substrate) were obtained in day-5 and day-27 rats but no changes were evident with regard to ADP:O ratios, respiratory control indices or the capacity for in vitro protein synthesis. By examination of subpopulations of mitochondria, it was found that mitochondria are heterogeneous with regard to ADP:O ratios, respiratory control indices, states 3 and 4 respiration and the capacity for in vitro protein synthesis. The heterogeneity for each of these parameters was altered in day-5 and day-27 animals. Although states 3 and 4 respiration were depressed throughout the entire mitochondrial population for day-5 and day-27 rats, a subpopulation of mitochondria from day-27 rats showed respiratory control indices and ADP:O ratios which were higher than any subpopulation of mitochondria of either day-5 or day-0 animals.     

Effects of phthalate esters on the respiration of rat liver mitochondria.

The effects of phthalate esters on the oxidation of succinate, glutamate, beta-hydroxybutyrate and NADH by rat liver mitochondria were examined and it was found that di-n-butyl phthalate (DBP) strongly inhibited the succinate oxidation by intact and sonicated rat mitochondria, but did not inhibit the State 4 respiration with NAD-linked substrates such as glutamate and beta-hydroxybutyrate. However, oxygen uptake accelerated by the presence of ADP and substrate (State 3) was inhibited and the rate of oxygen uptake decreased to that without ADP (State 4). It was concluded that phthalate esters were electron and energy transport inhibitors but not uncouplers. Phthalate esters also inhibited NADH oxidation by sonicated mitochondria. The degree of inhibition depended on the carbon number of alkyl groups of phthalate esters, and DBP was the most potent inhibitor of respiration. The activity of purified beef liver glutamate dehydrogenase [EC 1.4.1.3] was slightly inhibited by phthalate esters.     

Features of the Uncoupling Effect of Fatty Acids in Liver Mitochondria of Mammals with Different Body Weight

In the presence of oligomycin, EGTA, and magnesium ions, the protonophore uncoupling activity of palmitate (V(Pal)) is determined as the ratio of the acceleration of respiration with palmitate to its concentration. Under these conditions, V(Pal) in liver mitochondria of one-month-old rats with the body weight of 50 g is 1.46-fold higher than in liver mitochondria of adult rats with the body weight of 250 g, whereas the uncoupling activity of FCCP does not depend on the age of the animals. The difference in V(Pal) is mainly due to its component insensitive to carboxyatractylate and glutamate (V(Ins)). This value is 2.9-fold higher in mitochondria of one-month-old rats than in those of adult rats. The protonophore activity of palmitate is similar in liver mitochondria of four-day-old and adult rats. In liver mitochondria of adult mammals (mouse, rat, guinea pig, rabbit), V(Pal) decreases with increase in the body weight of the animals. In double logarithmic coordinates, the dependence of the V(Pal) value on the body weight is linear with slope angle tangent of -0.18. The V(Pal) value is mainly contributed by its component V(Ins). In the presence of calcium ions, palmitate induces the nonspecific permeability of the inner membrane of liver mitochondria (pore opening). This Ca2+-dependent uncoupling effect of palmitate is less pronounced in mitochondria of one-month-old rats than in those of adult rats. In mitochondria of adult animals (mice, rats, and guinea pigs), the Ca2+-dependent uncoupling activity of palmitate is virtually the same. It is concluded that the protonophore uncoupling effect of palmitate in liver mitochondria of mammals, unlike its Ca2+-dependent effect, is associated with thermogenesis at rest and also with production of additional heat on cooling of the animals.