Vitamin E-enriched diet reduces adaptive responses to training determining respiratory capacity and redox homeostasis in rat heart

We investigated whether reactive oxygen species (ROS) are involved in heart adaptive responses administering a vitamin E-enriched diet to trained rats. Using the homogenates and/or mitochondria from rat hearts we determined the aerobic capacity, tissue level of mitochondrial proteins, and expression of cytochrome c and factors (PGC-1, NRF-1, and NRF-2) involved in mitochondrial biogenesis. We also determined the oxidative damage, glutathione peroxidase (GPX) and reductase activities, glutathione content, mitochondrial ROS release rate, and susceptibility to in vitro oxidative challenge. Glutathione (GSH) content was not affected by both training and antioxidant supplementation. Conversely, antioxidant supplementation prevented metabolic adaptations to training, such as the increases in oxidative capacity, tissue content of mitochondrial proteins, and cytochrome c expression, attenuated some protective adaptations, such as the increase in antioxidant enzyme activities, and did not modify the decrease in ROS release by succinate supplemented mitochondria. Moreover, vitamin E prevented the training-linked increase in tissue capacity to oppose an oxidative attach. The antioxidant effects were associated with decreased levels of PGC-1, NRF-1, and NRF-2 expression. Our results support the idea that some heart adaptive responses to training depend on ROS produced during the exercise sessions and are mediated by the increase in PGC-1 expression which is involved in both the regulation of respiratory capacity and antioxidant protection. However, vitamin inability to prevent some adaptations suggests that other signaling pathways impinging on PGC-1 can modify the response to the antioxidant integration.     

Vitamin E supplementation modifies adaptive responses to training in rat skeletal muscle

Abstract

Aim of the present study was to test, by vitamin E treatment, the hypothesis that muscle adaptive responses to training are mediated by free radicals produced during the single exercise sessions. Therefore, we determined aerobic capacity of tissue homogenates and mitochondrial fractions, tissue content of mitochondrial proteins and expression of factors (PGC-1, NRF-1, and NRF-2) involved in mitochondrial biogenesis. Moreover, we determined the oxidative damage extent, antioxidant enzyme activities, and glutathione content in both tissue preparations, mitochondrial ROS production rate. Finally we tested mitochondrial ROS production rate and muscle susceptibility to oxidative stress. The metabolic adaptations to training, consisting in increased muscle oxidative capacity coupled with the proliferation of a mitochondrial population with decreased oxidative capacity, were generally prevented by antioxidant supplementation. Accordingly, the expression of the factors involved in mitochondrial biogenesis, which were increased by training, was restored to the control level by the antioxidant treatment. Even the training-induced increase in antioxidant enzyme activities, glutathione level and tissue capacity to oppose to an oxidative attach were prevented by vitamin E treatment. Our results support the idea that the stimulus for training-induced adaptive responses derives from the increased production, during the training sessions, of reactive oxygen species that stimulates the expression of PGC-1, which is involved in mitochondrial biogenesis and antioxidant enzymes expression. On the other hand, the observation that changes induced by training in some parameters are only attenuated by vitamin E treatment suggests that other signaling pathways, which are activated during exercise and impinge on PGC-1, can modify the response to the antioxidant integration.     

Acute administration of 3,5-diiodo-L-thyronine to hypothyroid rats stimulates bioenergetic parameters in liver mitochondria

The role of 3,5-diiodo-L-thyronine (T₂), initially considered only a 3,3′,5-triiodo-L-thyronine (T₃) catabolite, in the bioenergetic metabolism is of growing interest. In this study we investigated the acute effects (within 1 h) of T₂ administration to hypothyroid rats on liver mitochondria fatty acid uptake and β-oxidation rate, mitochondrial efficiency (by measuring proton leak) and mitochondrial oxidative damage (by determining H₂O₂ release). Fatty acid uptake into mitochondria was measured assaying carnitine palmitoyl transferase (CPT) I and II activities, and fatty acid β-oxidation using palmitoyl-CoA as a respiratory substrate. Mitochondrial fatty acid pattern was defined by gas-liquid chromatography. In hypothyroid + T₂ vs hypothyroid rats we observed a raise in the serum level of nonesterified fatty acids (NEFA), in the mitochondrial CPT system activity and in the fatty acid β-oxidation rate. A parallel increase in the respiratory chain activity, mainly from succinate, occurs. When fatty acids are chelated by bovine serum albumin, a T₂-induced increase in both state 3 and state 4 respiration is observed, while, when fatty acids are present, mitochondrial uncoupling occurs together with increased proton leak, responsible for mitochondrial thermogenesis. T₂ administration decreases mitochondrial oxidative stress as determined by lower H₂O₂ production. We conclude that in rat liver mitochondria T₂ acutely enhances the rate of fatty acid β-oxidation, and the activity of the downstream respiratory chain. The T₂-induced increase in proton leak may contribute to mitochondrial thermogenesis and to the reduction of oxidative stress. Our results strengthen the previously reported ability of T₂ to reduce adiposity, dyslipidemia and to prevent liver steatosis.     

Parthenolide regulates oxidative stress-induced mitophagy and suppresses apoptosis through p53 signaling pathway in C2C12 myoblasts

Muscle redox disturbances and oxidative stress have emerged as a common pathogenetic mechanism and potential therapeutic intervention in some muscle diseases. Parthenolide (PTL), a sesquiterpene lactone found in large amounts in the leaves of feverfew, possesses anti-inflammatory, anti-migraine, and anticancer properties. Although PTL was reported to alleviate cancer cachexia and improve skeletal muscle characteristics in a cancer cachexia model, its actions on oxidative stress-induced damage in C2C12 myoblasts have not been reported and the regulatory mechanisms have not yet been defined. In our study, PTL attenuated H₂O₂-induced growth inhibition and morphological changes. Furthermore, PTL exhibited scavenging activity against reactive oxygen species and protected C2C12 cells from apoptosis in response to H₂O₂. Meanwhile, PTL suppressed collapse of the mitochondrial membrane potential, thereby contributing to normalizing H₂O₂-induced autophagy flux and mitophagy, correlating with inhibiting degradation of mitochondrial marker protein TIM23, the increase in LC3-II expression and the reduction of mitochondria DNA. Besides its protective effect on mitochondria, PTL also prevented H₂O₂-induced lysosomes damage in C2C12 cells. In addition, the phosphorylation of p53, cathepsin B, and Bax/Bcl-2 protein levels, and the translocation of Bax from the cytosol to mitochondria induced by H₂O₂ in C2C12 cells was significantly reduced by PTL. In conclusion, PTL modulates oxidative stress-induced mitophagy and protects C2C12 myoblasts against apoptosis, suggesting a potential protective effect against oxidative stress-associated skeletal muscle diseases.     

Lung endothelial barrier protection by resveratrol involves inhibition of HMGB1 release and HMGB1-induced mitochondrial oxidative damage via an Nrf2-dependent mechanism

High-mobility group box 1 (HMGB1) contributes to lung vascular hyperpermeability during ventilator-induced lung injury. We aimed to determine whether the natural antioxidant resveratrol protected against HMGB1-induced endothelial hyperpermeability both in vitro and in vivo. We found that HMGB1 decreased vascular endothelial (VE)-cadherin expression and increased endothelial permeability, leading to mitochondrial oxidative damage in primary cultured mouse lung vascular endothelial cells (MLVECs). Both the mitochondrial superoxide dismutase 2 mimetic MnTBAP and resveratrol blocked HMGB1-induced mitochondrial oxidative damage, VE-cadherin downregulation, and endothelial hyperpermeability. In in vivo studies, anesthetized male ICR mice were ventilated for 4 h using low tidal volume (6 ml/kg) or high tidal volume (HV_T; 30 ml/kg) ventilation. The mice were injected intraperitoneally with resveratrol immediately before the onset of ventilation. We found that resveratrol attenuated HV_T-associated lung vascular hyperpermeability and HMGB1 production. HV_T caused a significant increase in nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation and Nrf2 target gene expression in lung tissues, which was further enhanced by resveratrol treatment. HMGB1 had no effect on Nrf2 activation, whereas resveratrol treatment activated the Nrf2 signaling pathway in HMGB1-treated MLVECs. Moreover, Nrf2 knockdown reversed the inhibitory effects of resveratrol on HMGB1-induced mitochondrial oxidative damage and endothelial hyperpermeability. The inhibitory effect of resveratrol on cyclic stretch-induced HMGB1 mRNA expression in primary cultured MLVECs was also abolished by Nrf2 knockdown. In summary, this study demonstrates that resveratrol protects against lung endothelial barrier dysfunction initiated by HV_T. Lung endothelial barrier protection by resveratrol involves inhibition of mechanical stretch-induced HMGB1 release and HMGB1-induced mitochondrial oxidative damage. These protective effects of resveratrol might be mediated through an Nrf2-dependent mechanism.     

Curcumin ameliorates CKD-induced mitochondrial dysfunction and oxidative stress through inhibiting GSK-3β activity

Curcumin has been reported to attenuate muscle atrophy. However, the underling mechanism remains unclear. The aim of this study was to investigate whether curcumin could improve chronic kidney disease (CKD)-induced muscle atrophy and mitochondrial dysfunction by inhibiting glycogen synthase kinase-3β (GSK-3β) activity. The sham and CKD mice were fed either a control diet or an identical diet containing 0.04% curcumin for 12 weeks. The C2C12 myotubes were treated with H₂O₂ in the presence or absence of curcumin. In addition, wild-type and muscle-specific GSK-3β knockout (KO) CKD model mice were made by 5/6 nephrectomy, and the sham was regarded as control. Curcumin could exert beneficial effects, including weight maintenance and improved muscle function, increased mitochondrial biogenesis, alleviated mitochondrial dysfunction by increasing adenosine triphosphate levels, activities of mitochondrial electron transport chain complexes and basal mitochondrial respiration and suppressing mitochondrial membrane potential. In addition, curcumin modulated redox homeostasis by increasing antioxidant activity and suppressed mitochondrial oxidative stress. Moreover, the protective effects of curcumin had been found to be mediated via inhibiting GSK-3β activity in vitro and in vivo. Importantly, GSK-3β KO contributed to improved mitochondrial function, attenuated mitochondrial oxidative damage and augmented mitochondrial biogenesis in muscle of CKD. Overall, this study suggested that curcumin alleviated CKD-induced mitochondrial oxidative damage and mitochondrial dysfunction via inhibiting GSK-3β activity in skeletal muscle.     

Differential effects of thyroid status on regional H2O2 production in slow- and fast-twitch muscle of ducklings

Birds seem to employ powerful physiological strategies to curb the harmful effects of reactive oxygen species (ROS) because they generally live longer than predicted by the free radical theory of aging. However, little is known about the physiological mechanisms that confer protection to birds against excessive ROS generation. Hence, we investigated the ability of birds to control mitochondrial ROS generation during physiologically stressful periods. In our study, we analyzed the relationship between the thyroid status and the function of intermyofibrillar and subsarcolemmal mitochondria located in glycolytic and oxidative muscles of ducklings. We found that the intermyofibrillar mitochondria of both glycolytic and oxidative muscles down regulate ROS production when plasma T₃ levels rise. The intermyofibrillar mitochondria of the gastrocnemius muscle (an oxidative muscle) produced less ROS and were more sensitive than the pectoralis muscle (a glycolytic muscle) to changes in plasma T₃. Such differences in the ROS production by glycolytic and oxidative muscles were associated with differences in the membrane proton permeability and in the rate of free radical leakage within the respiratory chain. This is the first evidence which shows that in birds, the amount of ROS that the mitochondria release is dependent on: (1) their location within the muscle; (2) the type of muscle (glycolytic or oxidative) and (3) on the thyroid status. Reducing muscle mitochondrial ROS generation might be an important mechanism in birds to limit oxidative damage during periods of physiological stress.     

Effect of thyroid state on susceptibility to oxidants and swelling of mitochondria from rat tissues

The effects of the thyroid state on oxidative damage, antioxidant capacity, susceptibility to in vitro oxidative stress and Ca(2+)-induced permeabilization of mitochondria from rat tissues (liver, heart, and gastrocnemious muscle) were examined. Hypothyroidism was induced by administering methimazole in drinking water for 15 d. Hyperthyroidism was elicited by a 10 d treatment of hypothyroid rats with triiodothyronine (10 micro g/100 g body weight). Mitochondrial levels of hydroperoxides and protein-bound carbonyls significantly decreased in hypothyroid tissues and were reported above euthroid values in hypothyroid rats after T(3) treatment. Mitochondrial vitamin E levels were not affected by changes of animal thyroid state. Mitochondrial Coenzyme Q9 levels decreased in liver and heart from hypothyroid rats and increased in all hyperthyroid tissues, while Coenzyme Q10 levels decreased in hypothyroid liver and increased in all hyperthyroid tissues. The antioxidant capacity of mitochondria was not significantly different in hypothyroid and euthyroid tissues, whereas it decreased in the hyperthyroid ones. Susceptibility to in vitro oxidative challenge decreased in mitochondria from hypothyroid tissues and increased in mitochondria from hyperthyroid tissues, while susceptibility to Ca(2+)-induced swelling decreased only in hypothyroid liver mitochondria and increased in mitochondria from all hyperthyroid tissues. The tissue-dependence of the mitochondrial susceptibility to stressful conditions in altered thyroid states can be explained by different thyroid hormone-induced changes in mitochondrial ROS production and relative amounts of mitochondrial hemoproteins and antioxidants. We suggest that susceptibilities to oxidants and Ca(2+)-induced swelling may have important implications for the thyroid hormone regulation of the turnover of proteins and whole mitochondria, respectively.     

H2O2 production and response to stress conditions by mitochondrial fractions from rat liver

Rat liver mitochondria, in different steps of the maturation process, were resolved by differential centrifugation at 1000g (M₁), 3000g (M₃), and 10,000g (M₁₀), and their characteristics determining susceptibility to stress conditions were investigated. Some parameters did not show gradual changes in the transition from M₁₀ to M₁ fraction because of the contamination of the M₁₀ fraction by microsomes and damaged mitochondria with relatively high lipid content. The highest and lowest rates of O₂ consumption and H₂O₂ production were exhibited by M₁ and M₁₀ fractions, respectively. Vitamin E and coenzyme Q levels were significantly higher in M₁₀ than in M₁ fraction, whereas whole antioxidant capacity was not significantly different. The degree of oxidative damage to lipids and proteins was higher in M₁ and not significantly different in M₃ and M₁₀ fractions. The order of susceptibility to both oxidative challenge and Ca²⁺-induced swelling was M₁ > M₃ > M₁₀. It seems that the Ca²⁺-induced swelling is due to permeabilization of oxidatively altered inner membrane and leads to discard mitochondria with high ROS production. If, as previous reports suggest, mitochondrial damage is initiating stimulus to mitochondrial biogenesis, the susceptibility of the M₁ mitochondria to stressful conditions could be important to regulate cellular ROS production. In fact, it should favor the substitution of the oldest ROS-overproducing mitochondria with neoformed mitochondria endowed with a smaller capacity to produce free radicals.     

Effect of voluntary exercise on H2O2 release by subsarcolemmal and intermyofibrillar mitochondria

Previous data have demonstrated that, to handle the oxidative stress encountered with training at high intensity, skeletal muscle relies on an increase in mitochondrial biogenesis, a reduced H(2)O(2) production, and an enhancement of antioxidant enzymes. In the present study, we evaluated the influence of voluntary running on mitochondrial O(2) consumption and H(2)O(2) production by intermyofibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) isolated from oxidative muscles in conjunction with the determination of antioxidant capacities. When mitochondria are incubated with succinate as substrate, both maximal (state 3) and resting (state 4) O(2) consumption were significantly lower in SSM than in IFM populations. Mitochondrial H(2)O(2) release per unit of O(2) consumed was 2-fold higher in SSM than in IFM. Inhibition of H(2)O(2) formation by rotenone suggests that complex I of the electron transport chain is likely the major physiological H(2)O(2)-generating system. In Lou/C rats (an inbred strain of rats of Wistar origin), neither O(2) consumption nor H(2)O(2) release by IFM and SSM were affected by long-term, voluntary wheel training. In contrast, glutathione peroxidase and catalase activity were significantly increased despite no change in oxidative capacities with long-term, voluntary exercise. Furthermore, chronic exercise enhanced heat shock protein 72 accumulation within skeletal muscle. It is concluded that the antioxidant status of muscle can be significantly improved by prolonged wheel exercise without necessitating an increase in mitochondrial oxidative capacities.     

Thyroid hormone induction of mitochondrial activity is coupled to mitophagy via ROS-AMPK-ULK1 signaling

Currently, there is limited understanding about hormonal regulation of mitochondrial turnover. Thyroid hormone (T₃) increases oxidative phosphorylation (OXPHOS), which generates reactive oxygen species (ROS) that damage mitochondria. However, the mechanism for maintenance of mitochondrial activity and quality control by this hormone is not known. Here, we used both in vitro and in vivo hepatic cell models to demonstrate that induction of mitophagy by T₃ is coupled to oxidative phosphorylation and ROS production. We show that T₃ induction of ROS activates CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, β) mediated phosphorylation of PRKAA1/AMPK (5′ AMP-activated protein kinase), which in turn phosphorylates ULK1 (unc-51 like autophagy activating kinase 1) leading to its mitochondrial recruitment and initiation of mitophagy. Furthermore, loss of ULK1 in T₃-treated cells impairs both mitophagy as well as OXPHOS without affecting T₃ induced general autophagy/lipophagy. These findings demonstrate a novel ROS-AMPK-ULK1 mechanism that couples T₃-induced mitochondrial turnover with activity, wherein mitophagy is necessary not only for removing damaged mitochondria but also for sustaining efficient OXPHOS.     

Adrenaline induces mitochondrial biogenesis in rat liver

We studied the effects of adrenaline administration and depletion (induced by reserpine) on rat liver oxidative metabolism. We showed that adrenaline increases, and reserpine decreases aerobic capacity (inferred by cytochrome oxidase activity) in tissue modifying the hepatic content of mitochondrial proteins without changing mitochondrial aerobic capacity. The changes in tissue cytochrome oxidase activity, which agreed with the expression levels of factors involved in mitochondrial biogenesis, such as PGC-1, NRF-1, and NRF-2, were associated with similar changes in tissue and mitochondrial State 3 respiration. Adrenaline and reserpine induced extensive lipid and protein oxidative damage in tissue and mitochondria. The increase in H₂O₂ release by respiring mitochondria and the decrease in the activities of the antioxidant enzymes glutathione peroxidase and reductase contributed to the reserpine effect on oxidative damage. The adrenaline effect is more difficult to explain, since the hormone increased the antioxidant enzyme activities but, in respiring mitochondria, increased ROS release rate in the presence of succinate and decreased it in the presence of pyruvate/malate. These opposite changes were due to the increased content of the autoxidizable electron carrier located at complex III and decreased content of that located at complex I. Our data suggest that adrenaline can be involved in the mitochondrial population adaptation which verify in conditions in which an increased body energy expenditure verify such as cold exposure.     

Moderate endurance training prevents doxorubicin-induced in vivo mitochondriopathy and reduces the development of cardiac apoptosis

The objective of this work was to test the hypothesis that endurance training may be protective against in vivo doxorubicin (DOX)-induced cardiomyopathy through mitochondria-mediated mechanisms. Forty adult (6-8 wk old) male Wistar rats were randomly divided into four groups (n = 10/group): nontrained, nontrained + DOX treatment (20 mg/kg), trained (14 wk of endurance treadmill running, 60-90 min/day), and trained + DOX treatment. Mitochondrial respiration, calcium tolerance, oxidative damage, heat shock proteins (HSPs), antioxidant enzyme activity, and apoptosis markers were evaluated. DOX induces mitochondrial respiratory dysfunction, oxidative damage, and histopathological lesions and triggers apoptosis (P < 0.05, n = 10). However, training limited the decrease in state 3 respiration, respiratory control ratio (RCR), uncoupled respiration, aconitase activity, and protein sulfhydryl content caused by DOX treatment and prevented the increased sensitivity to calcium in nontrained + DOX-treated rats (P < 0.05, n = 10). Moreover, training inhibited the DOX-induced increase in mitochondrial protein carbonyl groups, malondialdehyde, Bax, Bax-to-Bcl-2 ratio, and tissue caspase-3 activity (P < 0.05, n = 10). Training also increased by approximately 2-fold the expression of mitochondrial HSP-60 and tissue HSP-70 (P < 0.05, n = 10) and by approximately 1.5-fold the activity of mitochondrial and cytosolic forms of SOD (P < 0.05, n = 10). We conclude that endurance training protects heart mitochondrial respiratory function from the toxic effects of DOX, probably by improving mitochondrial and cell defense systems and reducing cell oxidative stress. In addition, endurance training limited the DOX-triggered apoptosis.     

Curcumin prevents Cr(VI)-induced renal oxidant damage by a mitochondrial pathway

We report the role of mitochondria in the protective effects of curcumin, a well-known direct and indirect antioxidant, against the renal oxidant damage induced by the hexavalent chromium [Cr(VI)] compound potassium dichromate (K₂Cr₂O₇) in rats. Curcumin was given daily by gavage using three different schemes: (1) complete treatment (100, 200, and 400 mg/kg bw 10 days before and 2 days after K₂Cr₂O₇ injection), (2) pretreatment (400 mg/kg bw for 10 days before K₂Cr₂O₇ injection), and (3) posttreatment (400 mg/kg bw 2 days after K₂Cr₂O₇ injection). Rats were sacrificed 48 h later after a single K₂Cr₂O₇ injection (15 mg/kg, sc) to evaluate renal and mitochondrial function and oxidant stress. Curcumin treatment (schemes 1 and 2) attenuated K₂Cr₂O₇-induced renal dysfunction, histological damage, oxidant stress, and the decrease in antioxidant enzyme activity both in kidney tissue and in mitochondria. Curcumin pretreatment attenuated K₂Cr₂O₇-induced mitochondrial dysfunction (alterations in oxygen consumption, ATP content, calcium retention, and mitochondrial membrane potential and decreased activity of complexes I, II, II-III, and V) but was unable to modify renal and mitochondrial Cr(VI) content or to chelate chromium. Curcumin posttreatment was unable to prevent K₂Cr₂O₇-induced renal dysfunction. In further experiments performed in curcumin (400 mg/kg)-pretreated rats it was found that this antioxidant accumulated in kidney and activated Nrf2 at the time when K₂Cr₂O₇ was injected, suggesting that both direct and indirect antioxidant effects are involved in the protective effects of curcumin. These findings suggest that the preservation of mitochondrial function plays a key role in the protective effects of curcumin pretreatment against K₂Cr₂O₇-induced renal oxidant damage.     

Human brain cortex: mitochondrial oxidative damage and adaptive response in Parkinson disease and in dementia with Lewy bodies

Frontal cortex samples from frozen human brains were used to assess tissue respiration; content of mitochondria; mitochondrial oxygen uptake; activity of respiratory complexes and of mitochondrial nitric oxide synthase (mtNOS); content of cytochromes a, b, and c; oxidative damage (protein carbonyls and TBARS); and expression of Mn-SOD in patients with Parkinson disease (PD) and with dementia with Lewy bodies (DLB) in comparison with those of normal healthy controls. Brain cortex and mitochondrial O₂ uptake and complex I activity were significantly lower in PD and DLB, whereas mtNOS activity, cytochrome content, expression of Mn-SOD, mitochondrial mass, and oxidative damage were significantly higher in the frontal cortex in PD and DLB. The decreases in tissue and mitochondrial O₂ uptake and in complex I activity are considered the consequences of mitochondrial oxidative damage. The increases in mtNOS activity and in mitochondrial mass are interpreted as an adaptive response of the frontal cortex that involves increased NO signaling for mitochondrial biogenesis. The adaptive response would partially compensate for mitochondrial dysfunction in these neurodegenerative diseases and would afford a human evolutionary response to shortage of ATP in the frontal cortex.     

Myoglobin causes oxidative stress,increase of NO production and dysfunction of kidney's mitochondria

Rhabdomyolysis or crush syndrome is a pathology caused by muscle injury resulting in acute renal failure. The latest data give strong evidence that this syndrome caused by accumulation of muscle breakdown products in the blood stream is associated with oxidative stress with primary role of mitochondria. In order to evaluate the significance of oxidative stress under rhabdomyolysis we explored the direct effect of myoglobin on renal tubules and isolated kidney mitochondria while measuring mitochondrial respiratory control, production of reactive oxygen and nitrogen species and lipid peroxidation. In parallel, we evaluated mitochondrial damage under myoglobinurea in vivo. An increase of lipid peroxidation products in kidney mitochondria and release of cytochrome c was detected on the first day of myoglobinuria. In mitochondria incubated with myoglobin we detected respiratory control drop, uncoupling of oxidative phosphorylation, an increase of lipid peroxidation products and stimulated NO synthesis. Mitochondrial pore inhibitor, cyclosporine A, mitochondria-targeted antioxidant (SkQ1) and deferoxamine (Fe-chelator and ferryl-myoglobin reducer) abrogated these events. Similar effects (oxidative stress and mitochondrial dysfunction) were revealed when myoglobin was added to isolated renal tubules. Thus, rhabdomyolysis can be considered as oxidative stress-mediated pathology with mitochondria to be the primary target and possibly the source of reactive oxygen and nitrogen species. We speculate that rhabdomyolysis-induced kidney damage involves direct interaction of myoglobin with mitochondria possibly resulting in iron ions release from myoglobin's heme, which promotes the peroxidation of mitochondrial membranes. Usage of mitochondrial permeability transition blockers, Fe-chelators or mitochondria-targeted antioxidants, may bring salvage from this pathology.     

3,5-Diiodo-L-Thyronine Administration To Hypothyroid Rats Rapidly Enhances Fatty Acid Oxidation Rate and Bioenergetic Parameters in Liver Cells

Growing evidence shows that, among triiodothyronine derivatives, 3,5 diiodo-L-thyronine (T₂) plays an important role in energy metabolism and fat storage. In the present study, short-term effects of T₂ administration to hypothyroid rats on fatty acid oxidation rate and bioenergetic parameters were investigated. Within 1 h following T₂ injection, state 3 and state 4 respiration rates, which were reduced in hypothyroid mitochondria, were noticeably increased particularly in succinate- with respect to glutamate/malate-energized mitochondria. Maximal respiratory activity, observed when glutamate/malate/succinate were simultaneously present in the respiratory medium, was significantly stimulated by T₂ treatment. A T₂-induced increase in respiratory rates was also observed when palmitoyl-CoA or L-palmitoylcarnitine were used as substrates. No significant change in respiratory control index and ADP/O ratio was observed. The activities of the mitochondrial respiratory chain complexes, especially Complex II, were increased in T₂-treated rats. In the latter, Complex V activities, assayed in both ATP synthesis and hydrolysis direction, were enhanced. The rate of fatty acid oxidation, followed by conversion of [¹⁴C]palmitate to CO₂ and ketone bodies, was higher in hepatocytes isolated from T₂-treated rats. This increase occurs in parallel with the raise in the activity of carnitine palmitoyltransferase-I, the rate limiting enzyme of fatty acid β-oxidation, assayed in situ in digitonin-permeabilized hepatocytes. Overall, these results indicate that T₂ rapidly increases the ability of mitochondria to import and oxidize fatty acids. An emerging idea in the literature is the ability of T₂ to reduce adiposity and dyslipidemia and to prevent the development in liver steatosis. The results of the present study, showing a rapid T₂-induced increase in the ability of mitochondria to import and oxidize fatty acids, may contribute to understand the biochemical mechanisms of T₂-metabolic effects.     

Neuroprotection of Chikusetsu saponin V on transient focal cerebral ischemia/reperfusion and the underlying mechanism

BackgroundOxidative stress and frequently unwanted alterations in mitochondrial structure and function are key aspects of the pathological cascade in transient focal cerebral ischemia. Chikusetsu saponin V (CHS V), a major component of saponins from Panax japonicas, can attenuate H₂O₂-induced oxidative stress in SH-SY5Y cells.PurposeThe aim of the present study was to investigate the neuroprotective effects and the possible underlying mechanism of CHS V on transient focal cerebral ischemia/reperfusion.MethodsMice with middle cerebral artery occlusion (MCAO) and cultured cortical neurons exposed to oxygen glucose deprivation (OGD) were used as in vivo and in vitro models of cerebral ischemia, respectively. The neurobehavioral scores, infarction volumes, H&E staining and some antioxidant levels in the brain were evaluated. The occurrence of neuronal death was estimated. Total and mitochondrial reactive oxygen species (ROS) levels, as well as mitochondrial potential were measured using flow cytometry analysis. Mitochondrial structure and respiratory activity were also examined. Protein levels were investigated by western blotting and immunohistochemistry.ResultsCHS V effectively attenuated cerebral ischemia/reperfusion (CI/R) injury, including improving neurological deficits, shrinking infarct volume and reducing the number of apoptotic cells. Furthermore, CHS V treatment remarkably increased antioxidant levels and reduced ROS levels and mitochondrial damage by enhancing the expression and deacetylation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) by activating AMPK and SIRT-1, respectively.ConclusionOur data demonstrated that CHS V prevented CI/R injury by suppressing oxidative stress and mitochondrial damage through the modulation of PGC-1α with AMPK and SIRT-1.     

Effect of cold-induced hyperthyroidism on H2O2 production and susceptibility to stress conditions of rat liver mitochondria

Previous studies have shown that T3 treatment and cold exposure induce similar biochemical changes predisposing rat liver to oxidative stress. This suggests that the liver oxidative damage observed in experimental and functional hyperthyroidism is mediated by thyroid hormone. To support this hypothesis we investigated whether middle-term cold exposure (2 and 10 days), like T3 treatment, also increases H2O2 release by liver mitochondria. We found that the rate of H2O2 release increased only during State 4 respiration, but faster flow of reactive oxygen species (ROS) from mitochondria to the cytosolic compartment was ensured by the concomitant increase in tissue mitochondrial proteins. Cold exposure also increased the capacity of mitochondria to remove H2O2. This indicates that cold causes accelerated H2O2 production, which might depend on enhanced autoxidizable carrier content and should lead to increased mitochondrial damage. Accordingly, mitochondrial levels of hydroperoxides and protein-bound carbonyls were higher after cold exposure. Levels of low-molecular weight antioxidants were not related to the extent of oxidative damage, but susceptibility to both in vitro oxidative challenge and Ca2+-induced swelling increased in mitochondria from cold exposed rats. The cold-induced changes in several parameters, including susceptibility to swelling, were time dependent, because they were apparent or greater after 10 days cold exposure. The cold-induced increase in swelling may be a feedback mechanism to limit tissue oxidative stress, purifying the mitochondrial population from ROS-overproducing mitochondria, and the time course for such change is consistent with the gradual development of cold adaptation.