Heat stress is of major concern for poultry, especially in the hot regions of the world because of the resulting poor growth performance, immunosuppression, and high mortality. To assess superoxide (O2*-) production in mitochondria isolated from skeletal muscle of chickens (n = 4 to 8) exposed to acute heat stress, electron spin resonance (ESR) spectroscopy using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap agent and lucigenin-derived chemiluminescence (LDCL) method were applied. ESR spectra of suspensions containing mitochondria from control and acute heat-treated meat-type chickens showed similar hyperfine coupling constants (aN = 1.44 mT, aHbeta = 0.12 mT, and aHbeta = 0.11 mT) to those of DMPO-O2*- adducts observed in a hypoxanthine-xanthine oxidase system. Heat exposure resulted in enhancement of the DMPO-O2*- signal. The results using LDCL showed significantly enhanced superoxide production in heat stress-treated skeletal muscle mitochondria of meat-type chickens, whereas no such increase was observed in laying chickens. The enhancement of superoxide production in the former case was associated with heat-induced increments in rectal and muscle temperatures, leading to significant body weight loss. In contrast, the latter case showed no increase in temperatures, although there was a slight decrease in body weight gain. Percentage increases of superoxide production in the presence of carboxyatractylate, a specific inhibitor of adenine nucleotide translocator (ANT), were the same for skeletal muscle mitochondria from meat- and laying-type chickens from the control or heat stress-treated group. This finding suggests the irrelevance of ANT in the regulation of reactive oxygen species flux under heat stress conditions. The study provides the first evidence of superoxide anion production in the skeletal muscle mitochondria of meat-type chickens in response to acute heat stress.

No abstract

Mitochondria have long been known to play a critical role in maintaining bioenergetic status under physiological conditions. Our previous studies have shown that acute heat stress increases production of mitochondrial reactive oxygen species (ROS) in the skeletal muscle of chickens. This increased ROS production may lead to nonspecific modification of lipids and proteins, which may then result in bioenergetic dysfunctions. If increased mitochondrial ROS production in heat-stressed chickens causes oxidative damage, changes in mitochondrial peroxidized lipids and oxidatively modified proteins can be detected. To study this, --week-old male broiler chickens (nῌ.ῌ2) were exposed to acute heat stress (-.῍ for +2 h) while control chickens were kept at thermoneutral condition (,/῍). Skeletal muscle subsarcolemmal mitochondria were isolated and used to study mitochondrial malondialdehyde (MDA) and protein carbonyl groups. Mitochondrial thiobarbituric acid reactive substances (TBARS) formation was measured colorimetrically with *.*+ῌ butylated hydroxytoluene (BHT) and expressed as MDA equivalent. To detect oxidation-sensitive mitochondrial proteins, polypeptides resolved by two-dimensional (,D) electrophoresis were immunostained with DNP-specific antibodies for carbonylated proteins using Western blotting. In heat-stressed chickens, mitochondrial MDA was ,.1-fold higher, and 2, mitochondrial proteins were oxidized when compared to that of control chickens. These results suggest that in heat-stressed chickens, increased mitochondrial ROS production leads to oxidative damage to mitochondrial lipids and proteins.

Acute heat stress (34 degrees C for 18 h) resulted in increased levels of reactive oxygen species (ROS) in mitochondria isolated from the skeletal muscle of broilers. This occurred when glutamate-requiring complexes I, III, and IV of the electron transport chain or succinate-requiring complexes II, III and IV were used as the substrate. This result confirms our previous observation that exposure of broilers to 34 degrees C for 18 h results in increased superoxide production in skeletal (pectoralis) muscle, and extends this finding by showing that substrate-independent ROS generation occurs during the heat stress period. When broilers were exposed to heat stress, the levels of avian uncoupling protein (avUCP) mRNA in skeletal muscle were significantly decreased, to 28% of the levels found in untreated controls. This was accompanied by a significant decrease in the levels of the avUCP protein, to 37% of control levels. In contrast, avian adenine nucleotide translocator mRNA levels were not affected by exposure to heat stress. This finding is consistent with previous studies which showed that the increases in superoxide production that are observed in the presence of carboxyatractylate, a specific inhibitor of adenine nucleotide translocator, were the same for skeletal muscle mitochondria from both control and heat-stressed chickens. Taken together, these results suggest that acute heat stress stimulates mitochondrial superoxide production in broiler skeletal muscle, possibly via downregulation of avUCP. The present study provides the first evidence that synthesis of avUCP protein is downregulated in heat-stressed broilers.

Although bird species studied thus far have no distinct brown adipose tissue (BAT) or a related thermogenic tissue, there is now strong evidence that non-shivering mechanisms in birds may play an important role during cold exposure. Recently, increased expression of the duckling homolog of the avian uncoupling protein (avUCP) was demonstrated in cold-acclimated ducklings [Raimbault et al., Biochem. J. 353 (2001) 441^444]. Among the mitochondrial anion carriers, roles for the ATP/ADP antiporter (ANT) as well as UCP variants in thermogenesis are proposed. The present experiments were conducted (i) to examine the e¡ects of cold acclimation on the fatty acid-induced uncoupling of oxidative phosphorylation in skeletal muscle mitochondria and (ii) to clone the cDNA of UCP and ANT homologs from chicken skeletal muscle and study di¡erences compared to controls in expression levels of their mRNAs in the skeletal muscle of cold-acclimated chickens. The results obtained here show that suppression of palmitate-induced uncoupling by carboxyatractylate was greater in the subsarcolemmal skeletal muscle mitochondria from cold-acclimated chickens than that for control birds. An increase in mRNA levels of avANT and, to lesser degree, of avUCP in the skeletal muscle of cold-acclimated chickens was also found. Taken together, the present studies on cold-acclimated chickens suggest that the simultaneous increments in levels of avANT and avUCP mRNA expression may be involved in the regulation of thermogenesis in skeletal muscle.

We have previously shown that avian uncoupling protein (avUCP) is downregulated on exposure to acute heat stress, stimulating mitochondrial reactive oxygen species (ROS) production and oxidative damage. In this study, we investigated whether upregulation of avUCP could attenuate oxidative damage caused by acute heat stress. Broiler chickens (Gallus gallus) were fed either a control diet or an olive oil-supplemented diet (6.7%), which has been shown to increase the expression of UCP3 in mammals, for 8 days and then exposed either to heat stress (34 degrees C, 12 h) or kept at a thermoneutral temperature (25 degrees C). Skeletal muscle mitochondrial ROS (measured as H(2)O(2)) production, avUCP expression, oxidative damage, mitochondrial membrane potential, and oxygen consumption were studied. We confirmed that heat stress increased mitochondrial ROS production and malondialdehyde levels and decreased the amount of avUCP. As expected, feeding birds an olive oil-supplemented diet increased the expression of avUCP in skeletal muscle mitochondria and decreased ROS production and oxidative damage. Studies on mitochondrial function showed that heat stress increased membrane potential in state 4, which was reversed by feeding birds an olive oil-supplemented diet, although no differences in basal proton leak were observed between control and heat-stressed groups. These results show that under heat stress, mitochondrial ROS production and olive oil-induced reduction of ROS production may occur due to changes in respiratory chain activity as well as avUCP expression in skeletal muscle mitochondria.

Mitochondrial dysfunction causes increased oxidative stress and depletion of ATP, which are involved in the etiology of a variety of renal diseases, such as CKD, AKI, and steroidresistant nephrotic syndrome. Antioxidant therapies are being investigated, but clinical outcomes have yet to be determined. Recently, we reported that a newly synthesized indole derivative, mitochonic acid 5 (MA-5), increases cellular ATP level and survival of fibroblasts from patients with mitochondrial disease. MA-5 modulates mitochondrial ATP synthesis independently of oxidative phosphorylation and the electron transport chain. Here, we further investigated the mechanism of action for MA-5. Administration of MA-5 to an ischemia-reperfusion injury model and a cisplatin-induced nephropathy model improved renal function. In in vitro bioenergetic studies, MA-5 facilitated ATP production and reduced the level of mitochondrial reactive oxygen species (ROS) without affecting activity of mitochondrial complexes I-IV. Additional assays revealed that MA-5 targets the mitochondrial protein mitofilin at the crista junction of the inner membrane. In Hep3B cells, overexpression of mitofilin increased the basal ATP level, and treatment with MA-5 amplified this effect. In a unique mitochondrial disease model (Mitomice with mitochondrial DNA deletion that mimics typical human mitochondrial disease phenotype), MA-5 improved the reduced cardiac and renal mitochondrial respiration and seemed to prolong survival, although statistical analysis of survival times could not be conducted. These results suggest that MA-5 functions in a manner differing from that of antioxidant therapy and could be a novel therapeutic drug for the treatment of cardiac and renal diseases associated with mitochondrial dysfunction.

Reactive oxygen species-induced damage of cells and molecules is one of the mechanisms responsible for the decline in an animal's performance due to heat stress. Mitochondria are the main producers of cellular superoxide, a process that is sensitive to proton motive force, and this superoxide production can be decreased by mild uncoupling. We studied the effects of heat stress on the production of mitochondrial superoxide as well as heat stress effects on the expression of avian uncoupling protein (avUCP) and avian A nucleotide translocator (avANT) in skeletal muscles of chicks and young cockerels. Male White Leghorn (Julia) chicks at 16 d and cockerels at 87 d of age were exposed to acute heat stress, 34 degrees C for 18 h, or kept at moderate ambient temperature (25 and 21 degrees C, respectively). There was no difference in mitochondrial superoxide production between heat-exposed and control chicks, whereas significant differences were observed in the case of young cockerels. Greater substrate-independent superoxide production was found in muscle mitochondria from heat-stressed young cockerels. In chicks, neither avUCP nor avANT transcript expression was changed by heat exposure, whereas in young cockerels avUCP transcript was decreased, but avANT transcript level was not changed. Thus, in heat-stressed young cockerels, increased mitochondrial superoxide production was accompanied by downregulation of avUCP. Taken together, these results suggest that exposure of young cockerels to heat stress stimulates mitochondrial superoxide production, possibly via downregulation of avUCP. Chicks with persistent avUCP expression, on the other hand, are relatively better adapted to high temperature. It can be assumed that appropriate expression of avUCP may alleviate overproduction of mitochondrial superoxide and could help birds adapt to oxidative stress resulting from acute heat stress.