Effect of melatonin treatment on oxygen consumption by rat liver mitochondria

Summary. The objective of this study was to examine the in vivo effect of melatonin on rat mitochondrial liver respiration. Two experiments were performed: For experiment 1, adult male rats received melatonin in the drinking water (16 or 50 μg/ml) or vehicle during 45 days. For experiment 2, rats received melatonin in the drinking water (50 μg/ml) for 45 days, or the same amount for 30 days followed by a 15 day-withdrawal period. At sacrifice, a liver mitochondrial fraction was prepared and oxygen consumption was measured polarographically in the presence of excess concentration of DL-3-β-hydroxybutyrate or L-succinate. Melatonin treatment decreased Krebs’ cycle substrate-induced respiration significantly at both examined doses. The stimulation of mitochondrial respiration caused by excess concentration of substrate recovered after melatonin withdrawal. Basal state 4 respiration was not modified by melatonin. Melatonin, by curtailing overstimulation of cellular respiration caused by excess Krebs’ cycle substrates, can protect the mitochondria from oxidative damage.     

Mitochondrial respiration in heart, liver, and kidney of copper-deficient rats

Morphological observations in some tissues indicate that dietary copper deficiency results in structural damage to mitochondria. The purpose of this study was to determine whether mitochondrial function is impaired as well. Male, weanling Sprague-Dawley rats were fed diets deficient or sufficient in copper for 4 weeks. Mitochondria were isolated from heart, liver, kidney cortex, and kidney medulla. P/O ratio, state 3 and state 4 respiration rates (oxygen consumed in the presence and absence of ADP, respectively), and acceptor control index (ratio of state 3:state 4) were determined using succinate or pyruvate/malate as substrate. State 3 respiration rate in mitochondria from copper-deficient hearts and livers was lower than in mitochondria from copper-sufficient hearts. Copper deficiency reduced the state 4 respiration rate only in cardiac mitochondria. Neither respiration rate was affected by copper deficiency in mitochondria from kidney medulla or cortex. P/O ratio was not significantly affected by copper deficiency in any tissue examined. Acceptor control index was reduced only in liver mitochondria. The observed decreases in respiration rates are consistent with decreased cytochrome c oxidase activity, shown by others to occur in mitochondria isolated from hearts and livers of copper-deficient rats.     

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.     

Effects of glucagon and 3,5,3'-triiodo-L-thyronine on oxidative phosphorylation of thyroidectomized rat liver mitochondria

In normal or thyroidectomized rat liver mitochondria, glucagon produced fast but transient stimulation of respiration rates in state 3 and state 4 whatever the substrates. Stimulation reached its maximum 20 to 30 minutes after glucagon injection. However, the effects of glucagon are less marked after removal of the thyroid gland, since the increases observed in the oxygen consumption and basal metabolic rates were only half those shown in normal rats. The activating effects of triiodothyronine and glucagon on the ADP phosphorylation rates were found to be additive. Pretreatment with cycloheximide blocked the activation induced by glucagon but not that induced by triiodothyronine. Both hormones therefore stimulate oxidative phosphorylation but by different mechanisms. Thyroidectomy did not alter the early rise in glycaemia observed in response to glucagon. It may therefore be assumed that the hypothyroid rat's sensitivity to glucagon is not directly connected with the change in cAMP metabolism.     

Mitochondrial superoxide and coenzyme Q in insulin-deficient rats: increased electron leak

Mitochondrial superoxide is important in the pathogeneses of diabetes and its complications. However, there is uncertainty regarding the intrinsic propensity of mitochondria to generate this radical. Studies to date suggest that superoxide production by mitochondria of insulin-sensitive target tissues of insulin-deficient rodents is reduced or unchanged. Moreover, little is known of the role of the Coenzyme Q (CoQ), whose semiquinone form reacts with molecular oxygen to generate superoxide. We measured reactive oxygen species (ROS) production, respiratory parameters, and CoQ content in mitochondria from gastrocnemius muscle of control and streptozotocin (STZ)-diabetic rats. CoQ content did not differ between mitochondria isolated from vehicle- or STZ-treated animals. CoQ also was unaffected by weight loss in the absence of diabetes (induced by caloric restriction). Under state 4 or state 3 conditions, both respiration and ROS release were reduced in diabetic mitochondria fueled with succinate, glutamate plus malate, or with all three substrates (continuous TCA cycle). However, H(2)O(2) and directly measured superoxide production were substantially increased in gastrocnemius mitochondria of diabetic rats when expressed per unit oxygen consumed. On the basis of substrate and inhibitor effects, the mechanism involved multiple electron transport sites. More limited results using heart mitochondria were similar. ROS per unit respiration was greater in muscle mitochondria from diabetic compared with control rats during state 3, as well as state 4, while the reduction in ROS per unit respiration on transition to state 3 was less for diabetic mitochondria. In summary, ROS production is, in fact, increased in mitochondria from insulin-deficient muscle when considered relative to electron transport. This is evident on multiple energy substrates and in different respiratory states. CoQ is not reduced in diabetic mitochondria or with weight loss due to food restriction. The implications of these findings are discussed.     

Oxidation of acetoacetate and palmitylcarnitine by brain and liver mitochondria from suckling and adult rats

Experiments in which we investigated the possible oxidative utilization of lipoid substrates by brain and liver mitochondria were carried out with rats aged 5 and 90 days, kept under completely standardized conditions. Brain mitochondria were isolated on a Ficoll gradient after Clark and Nicklas (1970). Respiratory activity (or the respiratory control index-R.C.) was determined in the manner described in an earlier paper (Dobesová and Mourek 1980). Na succinate or Na malate was used as the testing substrate; palmityl carnitine, acetyl carnitine and acetoacetate were used as lipoid substrates. Oxygen consumption was measured with a Clark's oxygen electrode and respiration was expressed in nAt oxygen per min per mg protein, which was measured by the method of Lowry et al. (1951). When using succinate or malate, in agreement with our previous results we did not find any development changes in the respiratory activity of the brain mitochondria. The oxidation of acetoacetate by the brain mitochondria of 5-day-old rats was about five times greater, and of acetyl carnitine over two times greater, compared with the CNS mitochondria of adult rats. The oxidative utilization of lipoid substrates by the liver mitochondria of 5-day-old rats was significantly greater than their utilization by CNS mitochondria (in the case of palmityl carnitine three times greater, for example) and was always significantly greater than in the liver mitochondria of adult rats. We demonstrated that mitochondria isolated from the brain of 5-day-old rats are equipped with an enzymatic apparatus which allows them to utilize lipoid substrates on a significantly greater scale than in adulthood.     

Age-associated deficit of mitochondrial oxidative phosphorylation in skeletal muscle: Role of carnitine and lipoic acid

Mitochondrial damage has implicated a major contributor for ageing process. In the present study, we measured mitochondrial membrane swelling, mitochondrial respiration (state 3 and 4) by using oxygen electrode in skeletal muscle of young (3–4 months old) and aged rats (above 24 months old) with supplementation of l-carnitine and dl-α-lipoic acid. Our results shows that the mitochondrial membrane swelling and state 4 respiration were increased more in skeletal muscle mitochondria of aged rats than in young control rats, whereas the state 3 respiration, respiratory control ratio (RCR) and ADP:O ratio decreased more in aged rats than in young rats. After supplementation of carnitine and lipoic acid to aged rats for 30 days, the state 3 respiration and RCR were increased, whereas the state 4 and mitochondrial membrane swelling were decreased to near normal rats. From our results, we conclude that combined supplementation of carnitine and lipoic acids to aged rats increases the skeletal muscle mitochondrial respiration, thereby increasing the level of ATP. (Mol Cell Biochem xxx: 83–89, 2005)     

Functional aspects of oxidative phosphorylation and electron transport in cardiac mitochondria of copper-deficient rats

Although dietary copper deficiency causes physiological, morphological, and biochemical abnormalities in cardiac mitochondria, the relationship observed between abnormalities of mitochondrial structure and function have been inconsistent in previous studies. The purpose of the present study was to re-evaluate the respiration rates of cardiac mitochondria from copper-deficient rats and to use several drugs that uncouple and inhibit mitochondrial respiration in order to clarify the mechanisms of mitochondrial dysfunction found in several laboratories. Copper deficiency reduced state 4 and state 3 cardiac mitochondrial respiration rates with all substrates tested. However, neither the ratio of ADP/oxygen consumed nor the acceptor control index was affected by copper deficiency. Cardiac mitochondria of copper-deficient rats showed a resistance to respiratory blockade by oligomycin and an increased ability to hydrolyze ATP in the presence of oligomycin compared with mitochondria of copper-adequate rats. This suggests that copper deficiency affects the function of the cardiac mitochondrial ATP synthase.     

Supplementation of curcumin and vitamin E enhances oxidative stress,but restores hepatic histoarchitecture in hypothyroid rats

AimsIn the present study, the effects of vitamin E and curcumin on hepatic dysfunction, mitochondrial oxygen consumption as well as hyperlipidemia in hypothyroid rats are reported.Main methodsAdult male rats were rendered hypothyroid by administration of 0.05% 6-n-propyl-2-thiouracil (PTU) in their drinking water, while vitamin E (200 mg/kg body weight) and curcumin (30 mg/kg body weight) were supplemented orally for 30 days.Key findingsHypothyroidism-induced elevation in serum aspartate aminotransferase activity was found to decline in vitamin E and curcumin treated rats. Nevertheless, distorted histoarchitecture revealed in hypothyroid rat liver was alleviated to normal by vitamin E and curcumin treatment. Regulation of hypothyroidism induced decrease in complexes I and II mediated mitochondrial respiration by vitamin E and curcumin was found to be different. Administration of curcumin to hypothyroid rats alleviates the decreased state 4 respiration and increased respiratory control ratio (RCR) level in complex I mediated mitochondrial oxygen consumption, whereas complex II mediated respiration was not influenced by exogenous antioxidants. Although, increase in serum concentration of total cholesterol was not modified by exogenous antioxidants, increased level of non-high-density lipoprotein cholesterol (non-HDL-C) in serum of hypothyroid rats was further enhanced by vitamin E and curcumin. Moreover, a significant elevation in mitochondrial lipid peroxidation and protein carbonylation was noticed in hypothyroid groups treated with vitamin E and curcumin.SignificanceThe present study suggests that supplementation of curcumin and vitamin E enhances oxidative stress parameters and hyperlipidemia; nevertheless, it protects hypothyroid-induced altered rectal temperature, serum transaminase activity and hepatic histoarchitecture.     

3,5-Diiodo-L-Thyronine Activates Brown Adipose Tissue Thermogenesis in Hypothyroid Rats

3,5-diiodo-l-thyronine (T2), a thyroid hormone derivative, is capable of increasing energy expenditure, as well as preventing high fat diet-induced overweight and related metabolic dysfunction. Most studies to date on T2 have been carried out on liver and skeletal muscle. Considering the role of brown adipose tissue (BAT) in energy and metabolic homeostasis, we explored whether T2 could activate BAT thermogenesis. Using euthyroid, hypothyroid, and T2-treated hypothyroid rats (all maintained at thermoneutrality) in morphological and functional studies, we found that hypothyroidism suppresses the maximal oxidative capacity of BAT and thermogenesis, as revealed by reduced mitochondrial content and respiration, enlarged cells and lipid droplets, and increased number of unilocular cells within the tissue. In vivo administration of T2 to hypothyroid rats activated BAT thermogenesis and increased the sympathetic innervation and vascularization of tissue. Likewise, T2 increased BAT oxidative capacity in vitro when added to BAT homogenates from hypothyroid rats. In vivo administration of T2 to hypothyroid rats enhanced mitochondrial respiration. Moreover, UCP1 seems to be a molecular determinant underlying the effect of T2 on mitochondrial thermogenesis. In fact, inhibition of mitochondrial respiration by GDP and its reactivation by fatty acids were greater in mitochondria from T2-treated hypothyroid rats than untreated hypothyroid rats. In vivo administration of T2 led to an increase in PGC-1α protein levels in nuclei (transient) and mitochondria (longer lasting), suggesting a coordinate effect of T2 in these organelles that ultimately promotes net activation of mitochondrial biogenesis and BAT thermogenesis. The effect of T2 on PGC-1α is similar to that elicited by triiodothyronine. As a whole, the data reported here indicate T2 is a thyroid hormone derivative able to activate BAT thermogenesis.     

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.     

Bioenergetics in aging: mitochondrial proton leak in aging rat liver, kidney and heart

Aging is associated with a decline in performance in many organs and loss of physiological performance can be due to free radicals. Mitochondria are incompletely coupled: during oxidative phosphorylation some of the redox energy is dissipated as natural proton leak across the inner membrane. To verify whether proton leak occurs in mitochondria during aging, we measured the mitochondrial respiratory chain activity, membrane potential and proton leak in liver, kidneys and heart of young and old rats. Mitochondria from old rats showed normal rates of Complex I and Complex II respiration. However, they had a lower membrane potential compared to mitochondria from younger rats. In addition, they exhibited an increased rate of proton conductance which partially dissipated the mitochondrial membrane potential when the rate of electron transport was suppressed. This could compromise energy homeostasis in aging cells in conditions that require additional energy supply and could minimize oxidative damage to DNA.     

Does high-sucrose diet alter skeletal muscle and liver mitochondrial respiration?

A diet high in sucrose or fructose progressively impairs glucose and lipid metabolism, which leads to insulin resistance. As mitochondria are the sites of the oxidation and utilization of these substrates, we hypothesized that a high sucrose diet would alter mitochondrial respiration. Male Wistar rats were fed high-sucrose (SU) or control (CTL) diet for one week; mitochondrial respiration was investigated in mitochondria isolated from liver and both glycolytic and oxidative muscles, with pyruvate and palmitate as substrates. To test for metabolic disturbances, we measured not only glycogen content in muscles and liver, but also lactate, glucose and triglyceride blood concentrations. After one week of high-sucrose intake, we found no change in blood concentration of these variables, but glycogen content was significantly increased in liver (17.28 +/- 2.98 mg/g tissue SU vs 6.47 +/- 1.67 mg/g tissue CTL), oxidative muscle (1.59 +/- 0.21 mg/g tissue SU vs 0.70 +/- 0.24 mg/g tissue CTL) though not in glycolytic muscle (1.72 +/- 0.44 mg/g tissue SU vs 1.52 +/- 0.20 mg/g tissue CTL). State 3 mitochondrial respiration was significantly decreased in SU rats compared with CTL (p < 0.05) with pyruvate, while no change was observed with palmitate. This study shows that 1-week of high-sucrose diet altered mitochondrial pyruvate oxidation in rats and suggests that, in the context of a high-sucrose diet, impaired mitochondrial respiration could contributed to the development of insulin resistance.     

Cadmium toxicity and liver mitochondria. I. Different effects of cadmium administered in vivo to adult, young, and ethionine-fed rats

The changes in liver mitochondrial respiratory activities and cytochrome concentrations were investigated when cadmium chloride was administered orally to adult, young, and ethionine-fed rats. Following a seven-day administration of 30 ppm cadmium in drinking water, adult rats showed no change, while young rats and ethionine-fed rats exhibited a marked increase in mitochondrial respiration with concomitant decrease of respiratory control index and P/O ratio. The concentrations of cytochromes aa3, b, and c + c1 in liver mitochondria were unchanged in adult rats, but increased significantly in ethionine-fed rats. In young rats receiving cadmium the liver mitochondrial protein increased with a slight change in the cytochrome concentration in mitochondria. It was further found that in adult rats a higher concentration (300 ppm) of cadmium in drinking water was toxic to the liver mitochondrial functions. Thus, the effect of oral administration of cadmium on the liver mitochondrial function depends on the condition of the animals.     

Ischemia-induced alterations of mitochondrial structure and function in brain, liver, and heart muscle of young and senescent rats

Young and senescent rats (3 and 28-30 months old) were subjected to complete ischemia at 37 degrees C in order to study function and structure of mitochondria isolated from liver, heart muscle, and brain. The rates of energy-coupled respiration and ATP synthesis were found to decrease progressively in relation to time of ischemia. The respiratory rates in the absence of ADP (state 4 respiration) did not increase after exposure to ischemia, suggesting that ischemia primarily affects electron transport rather than the energy coupling system. Mitochondria of heart muscle were more affected by ischemia than mitochondria of brain and liver. Liver and heart muscle mitochondria obtained from young rats were found to be slightly more sensitive to short periods of ischemia than those isolated from senescent animals.     

Adenine nucleotides, glutamate and respiratory function of heart mitochondria during acute hypoxia

The effect of acute hypoxia on adenine nucleotides, glutamate, aspartate, alanine and respiration of heart mitochondria was studied in rats. The losses of intramitochondrial adenine nucleotides (ATP+ADP+AMP) during hypoxia were related to depression of state 3 respiration supported by glutamate and malate, as well as decrease in uncoupled respiration. Hypoxia had less prominent effect on succinate-dependent state 3 respiration. Non-phosphorylating (state 4) respiratory rates and ADP/O ratios were slightly affected by oxygen deprivation. Glutamate fall in tissue and mitochondria of hypoxic hearts was concomitant with significant increase in tissue alanine and mitochondrial aspartate. The losses of intramitochondrial ATP and respiratory activity with NAD-dependent substrates during hypoxia were related to a decrease in mitochondrial glutamate. The results suggest that hypoxia-induced impairment of complex I of respiratory chain and a loss of glutamate from the matrix may limit energy-producing capacity of heart mitochondria.     

Effects of fasting on oxidative stress in rat liver mitochondria

While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O₂ consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (N^epsilon- malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.     

Rat liver response elicited by long-term cold exposure.

We measured mitochondrial protein mass as well as State 4 and 3 respiratory rates using different substrates in isolated liver mitochondria from 30-day cold-exposed rats. In addition, we measured the respiration under different conditions of stimulation in isolated hepatocytes from long-term cold-exposed rats. The results show that long-term cold exposure elicits a significant increase in hepatic mass and mitochondrial protein mass. No variation was found in oxygen consumption of isolated mitochondria and hepatocytes. On the whole, the results indicate that long-term exposure elicits an increase in hepatic mitochondrial protein mass but not in hepatic oxygen consumption.     

ATP Accelerates Respiration of Mitochondria From Rat Lung and Suppresses Their Release of Hydrogen Peroxide

Lung mitochondria were isolated by differential centrifugation from pentobarbital-anesthetized male rats. One to three millimolar Mg²⁺-ATP increased the consumption of oxygen of lung mitochondria oxidizing 10 mM succinate > fourfold (P < 0.01) whereas ATP increased the respiration of liver mitochondria by < 35%. ATP also hyperpolarized partially uncoupled lung mitochondria in the presence of the mitochondria-specific antagonist, oligomycin. However, only 20% of the ATPase activity in the lung mitochondria was blocked by oligomycin compared to a blockade of 91% for liver mitochondria. We investigated the effect of reducing the non-mitochondrial ATPase activity in the lung preparation. A purer suspension of lung mitochondria from a Percoll gradient was inhibited 95% by oligomycin. The volume fraction identified as mitochondria by electron microscopy in this suspension (73.6± 3.5%) did not differ from that for liver mitochondria (69.1± 4.9%). ATP reduced the mean area of the mitochondrial profiles in this Percoll fraction by 15% (P <0.01) and increased its state 3 respiration with succinate as substrate by 1.5-fold (P < 0.01) with no change in the state 4 respiration measured after carboxyatractyloside. Hence, ATP increased the respiratory control ratio (state 3/state 4, P <0.01). In contrast, state 3 respiration with the complex 1-selective substrates, glutamate and malate, did not change with addition of ATP. The acceleration of respiration by ATP was accompanied by decreased production of H₂O₂. Thus ATP-dependent processes that increase respiration appear to improve lung mitochondrial function while minimizing the release of reactive oxygen species.     

Peculiarities of tricarboxylic acid cycle in the myocardium of adult and old rats in the case of immobilization stress

Indices showing Krebs cycle functioning in the hearts of adult and old rats subjected to 30-minutes immobilization were studied in order to find out the causes of age-related increase of heart sensitivity to stress. The studies have shown that compensatory changes of energy metabolism promoting limitation of stress-induced decrease of energy supply of the heart muscle took place in the myocardium mitochondria in adult and old rats during immobilization stress. The changes are associated with maintenance of high rate of redox reactions in the Krebs cycle and increase of myocardium respiration in old rats, and with an increase of the FAD-dependent processes in tissue respiration in adult rats.