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International audience; The worldwide expansion of artificial light at night (ALAN) is acknowledged as a threat to biodiversity through alterations of the natural photoperiod triggering the disruption of physiological functions. In vertebrates, melatonin production during the dark phase can be decreased or suppressed by nocturnal light as shown in many taxa. But the effect of ALAN at low intensity mimicking light pollution in peri-urban area has never been investigated in amphibians. We filled this gap by studying the impact of low ALAN levels on the expression of genes related to melatonin synthesis and signaling in two anurans (agile frog, Rana dalmatina, and common toad, Bufo bufo). Circadian expression of genes encoding enzymes catalyzing melatonin synthesis (aralkylamine N- acetyltransferase, AANAT and acetylserotonin O-methyltransferase, ASMT) or melatonin receptors (Mel1a, Mel1b and Mel1c) was investigated using RT-qPCR after 23 days of nocturnal exposure to control (< 0.01 lx) or low ALAN (3 lx). We showed that the relative abundance of most transcripts was low in late afternoon and early evening (06 pm and 08 pm) and increased throughout the night in R. dalmatina. However, a clear and ample nocturnal pattern of target gene expression was not detected in control tadpoles of both species. Surprisingly, a low ALAN level had little influence on the relative expression of most melatonin-related genes. Only Mel1c expression in R. dalmatina and Mel1b expression in B. bufo were affected by ALAN. This target gene approach provides experimental evidence that melatonin signaling pathway was slightly affected by low ALAN level in anuran tadpoles.

International audience; Artificial light at night (ALAN) affects numerous physiological and behavioural mechanisms in various species by po- tentially disturbing circadian timekeeping systems and modifying melatonin levels. However, given the multiple direct and indirect effects of ALAN on organisms, large-scale transcriptomic approaches are essential to assess the global ef- fect of ALAN on biological processes. Moreover, although studies have focused mainly on variations in gene expression during the night in the presence of ALAN, it is necessary to investigate the effect of ALAN on gene expression during the day. In this study, we combined de novo transcriptome sequencing and assembly, and a controlled laboratory experi- ment to evaluate the transcriptome-wide gene expression response using high-throughput (RNA-seq) in Bufo bufo tad- poles exposed to ecologically relevant light levels. Here, we demonstrated for the first time that ALAN affected gene expression at night (3.5% and 11% of differentially expressed genes when exposed to 0.1 and 5 lx compared to con- trols, respectively), but also during the day (11.2% of differentially expressed genes when exposed to 5 lx compared to controls) with a dose-dependent effect. ALAN globally induced a downregulation of genes (during the night, 58% and 62% of the genes were downregulated when exposed to 0.1 and 5 lx compared to controls, respectively, and dur- ing the day, 61.2% of the genes were downregulated when exposed to 5 lx compared to controls). ALAN effects were detected at very low levels of illuminance (0.1 lx) and affected mainly genes related to the innate immune system and, to a lesser extend to lipid metabolism. These results provide new insights into understanding the effects of ALAN on organism. ALAN impacted the expression of genes linked to a broad range of physiological pathways at very low levels of ALAN during night-time and during daytime, potentially resulting in reduced immune capacity under environmen- tal immune challenges.

International audience; At fledging, juvenile king penguins (Aptenodytes patagonicus) must overcome the tremendous energetic constraints imposed by their marine habitat, including during sustained extensive swimming activity and deep dives in cold seawater. Both endurance swimming and skeletal muscle thermogenesis require high mitochondrial respiratory capacity while the submerged part of dive cycles repeatedly and greatly reduces oxygen availability, imposing a need for solutions to conserve oxygen. The aim of the present study was to determine in vitro whether skeletal muscle mitochondria become more ‘thermogenic’ to sustain heat production or more ‘economical’ to conserve oxygen in sea-acclimatized immature penguins (hereafter ‘immatures’) compared with terrestrial juveniles. Rates of mitochondrial oxidative phosphorylation were measured in permeabilized fibers and mitochondria from the pectoralis muscle. Mitochondrial ATP synthesis and coupling efficiency were measured in isolated muscle mitochondria. The mitochondrial activities of respiratory chain complexes and citrate synthase were also assessed. The results showed that respiration, ATP synthesis and respiratory chain complex activities in pectoralis muscles were increased by sea acclimatization. Furthermore, muscle mitochondria were on average 30–45% more energy efficient in sea-acclimatized immatures than in pre-fledging juveniles, depending on the respiratory substrate used (pyruvate, palmitoylcarnitine). Hence sea acclimatization favors the development of economical management of oxygen, decreasing the oxygen needed to produce a given amount of ATP. This mitochondrial phenotype may improve dive performance during the early marine life of king penguins, by extending their aerobic dive limit.

International audience; Mass-specific metabolic rate negatively co-varies with body mass from the whole-animal to the mitochondrial levels. Mitochondria are the mainly consumers of oxygen inspired by mammals to generate ATP or compensate for energetic losses dissipated as the form of heat ( proton leak) during oxidative phosphorylation. Consequently, ATP synthesis and proton leak compete for the same electrochemical gradient. Because proton leak co-varies negatively with body mass, it is unknown whether extremely small mammals further decouple their mitochondria to maintain their body temperature or whether they implement metabolic innovations to ensure cellular homeostasis. The present study investigated the impact of body mass variation on cellular and mitochondrial functioning in small mammals, comparing two extremely small African pygmy mice (Mus mattheyi, similar to 5 g, and Mus minutoides, similar to 7 g) with the larger house mouse (Mus musculus, similar to 22 g). Oxygen consumption rates were measured from the animal to the mitochondrial levels. We also measured mitochondrial ATP synthesis in order to appreciate the mitochondrial efficiency (ATP/O). At the whole-animal scale, mass- and surface-specific metabolic rates co-varied negatively with body mass, whereas this was not necessarily the case at the cellular and mitochondrial levels. Mus mattheyi had generally the lowest cellular and mitochondrial fluxes, depending on the tissue considered (liver or skeletal muscle), as well as having more-efficient muscle mitochondria than the other two species. Mus mattheyi presents metabolic innovations to ensure its homeostasis, by generating more ATP per oxygen consumed.

International audience; t fledging, king penguin juveniles undergo a major energetic challenge to overcome the intense and prolonged energy demands for thermoregulation and locomotion imposed by life in cold seas. Among other responses, sea acclimatization triggers fuel selection in skeletal muscle metabolism towards lipid oxidation in vitro, which is reflected by a drastic increase in lipid-induced thermogenesis in vivo. However, the exact nature of skeletal muscle thermogenic mechanisms (shivering and/or non-shivering thermogenesis) remains undefined. The aim of the present study was to determine in vivo whether the capacity for non-shivering thermogenesis was enhanced by sea acclimatization. We measured body temperature, metabolic rate, heart rate and shivering activity in fully immersed king penguins (Aptenodytes patagonicus) exposed to water temperatures ranging from 12 to 29°C. Results from terrestrial pre-fledging juveniles were compared with those from sea-acclimatized immature penguins (hereafter ‘immatures’). The capacity for thermogenesis in water was as effective in juveniles as in immatures, while the capacity for non- shivering thermogenesis was not reinforced by sea acclimatization. This result suggests that king penguins mainly rely on skeletal muscle contraction (shivering or locomotor activity) to maintain endothermy at sea. Sea-acclimatized immature penguins also exhibited higher shivering efficiency and oxygen pulse (amount of oxygen consumed or energy expended per heartbeat) than pre-fledging juvenile birds. Such increase in shivering and cardiovascular efficiency may favor a more efficient activity–thermoregulatory heat substitution providing penguins with the aptitude to survive the tremendous energetic challenge imposed by marine life in cold circumpolar oceans.

International audience; The mitochondrial genome (mt-DNA) functional repertoire has recently been enriched in mammals by the identification of functional small open reading frames (sORFs) embedded in ribosomal DNAs. Through com- parative genomic analyses the presence of putatively functional sORFs was investigated in birds. Alignment of available avian mt-DNA sequences revealed highly conserved regions containing four putative sORFs that pre- sented low insertion/deletion polymorphism rate (< 0.1%) and preserved in frame start/stop codons in > 80% of species. Detected sORFs included avian homologs of human Humanin and Short-Humanin-Like-Peptide 6 and two new sORFs not yet described in mammals. The amino-acid sequences of the four putative encoded peptides were strongly conserved among birds, with amino-acid p-distances (5.6 to 25.4%) similar to those calculated for typical avian mt-DNA-encoded proteins (14.8%). Conservation resulted from either drastic conservation of the nucleotide sequence or negative selection pressure. These data extend to birds the possibility that mitochondrial rDNA may encode small bioactive peptides.

International audience; Artificial Light At Night (ALAN) is an emerging pollution, that dramatically keeps on increasing worldwide due to urbanisation and transport infrastructure development. In 2016, it nearly affected 23% of the Earth’s surface. To date, all terrestrial and aquatic ecosystems have been affected. The disruption of natural light cycles due to ALAN is particularly expected for nocturnal species, which require dark periods to forage, move, and reproduce. Apart from chiropterans, amphibians contain the largest proportion of nocturnal species among vertebrates exhibiting an unfavourable conservation status in most parts of the world and living in ALAN polluted areas. Despite the growing number of studies on this subject, our knowledge on the direct influence of nocturnal lighting on amphibians is still scarce. To better understand the consequences of ALAN on the breeding component of amphibian fitness, we experimentally exposed male breeding common toads (Bufo bufo) to ecologically relevant light intensities of 0.01 (control), 0.1 or 5 lux for 12 days. At mating, exposed males took longer than controls to form an amplexus, i.e. to pair with a female, and broke amplexus before egg laying, while controls never did. These behavioural changes were associated with fitness alteration. The fertilisation rate of 5 lux-exposed males was reduced by 25%. Salivary testosterone, which is usually correlated with reproductive behaviours, was not altered by ALAN. Our study demonstrates that ALAN can affect the breeding behaviour of anuran species and reduce one component of their fitness. Given the growing importance of ALAN, more work is needed to understand its long-term consequences on the behaviour and physiology of individuals. It appears essential to identify deleterious effects for animal populations and propose appropriate management solutions in an increasingly brighter world.

International audience; Under nutritional deprivation, the energetic benefits of reducing mitochondrial metabolism are often associated with enhanced harmful pro-oxidant effects and a subsequent long-term negative impact on cellular integrity. However, the flexibility of mitochondrial functioning under stress has rarely been explored during the transition from basal non-phosphorylating to maximal phosphorylating oxygen consumption. Here, we experimentally tested whether ducklings (Cairina moscnata), fasted for 6 days and subsequently refed for 3 days, exhibited modifications to their mitochondrial fluxes, i.e. oxygen consumption, ATP synthesis, reactive oxygen species generation (ROS) and associated ratios, such as the electron leak (% ROS/O) and the oxidative cost of ATP production (% ROS/ATP). This was carried out at different steady-state rates of oxidative phosphorylation in both pectoralis (glycolytic) and gastrocnemius (oxidative) muscles. Fasting induced a decrease in the rates of oxidative phosphorylation and maximal ROS release. These changes were completely reversed by 3 days of refeeding. Yet, the fundamental finding of the present study was the existence of a clear threshold in ROS release and associated ratios, which remained low until a low level of mitochondrial activity was reached (30-40% of maximal oxidative phosphorylation activity).

Under nutritional deprivation, the energetic benefits of reducing mitochondrial metabolism are often associated with enhanced harmful pro-oxidant effects and a subsequent long-term negative impact on cellular integrity. However, the flexibility of mitochondrial functioning under stress has rarely been explored during the transition from basal non-phosphorylating to maximal phosphorylating oxygen consumption. Here, we experimentally tested whether ducklings (

Massive bubble formation after diving can lead to decompression sickness (DCS). Gut fermentation at the time of a dive exacerbates DCS due to endogenous hydrogen production. We sought to investigate whether medium-term stimulation of fermentation as a result of polyethylene glycol (PEG)-induced acceleration of bowel transit before diving exacerbates DCS in rats. Seven days before an experimental dry dive, 60 rats were randomly divided in two groups: an experimental group treated with PEG (n = 30) and an untreated control group (n = 30). Exhaled hydrogen was measured before the dive. Following hyperbaric exposure, we assessed for signs of DCS. After anaesthetisation, arterial blood was drawn to assay inflammatory cytokines and markers of oxidative stress. PEG led to a significant increase in exhaled H2 (35 ppm [10–73] compared with control 7 ppm [2–15]; p = 0.001). The probability of death was reduced in PEG-treated rats (PEG: 17% [95% CI 4–41] vs control: 50% [95% CI 26–74]; p = 0.034). In addition, inflammatory markers were reduced, and the antioxidant activity of glutathione peroxidase was significantly increased (529.2 U.l−1 [485.4–569.0] versus 366.4 U.l−1 [317.6–414.8]; p = 0.004). Thus, gut fermentation might have a positive effect on DCS. The antioxidant and neuroprotective properties of the fermentation by-products H2 and butyrate may explain these results.

International audience; In king penguin juveniles, the environmental transition from a terrestrial to a marine habitat, occurring at fledging, drastically stimulates lipid catabolism and the remodelling of muscle mitochondria to sustain extensive swimming activity and thermoregulation in the cold circumpolar oceans. However, the exact nature of these mechanisms remains only partially resolved. Here we investigated, in vitro, the uncoupling effect of increasing doses of fatty acids in pectoralis muscle intermyofibrillar mitochondria isolated, either from terrestrial never-immersed or experimentally cold water immersed pre-fledging king penguins or from sea-acclimatized fledged penguins. Mitochondria exhibited much greater palmitate-induced uncoupling respiration and higher maximal oxidative capacity after acclimatization to marine life. Such effects were not reproduced experimentally after repeated immersions in cold water, suggesting that the plasticity of mitochondrial characteristics may not be primarily driven by cold exposure per se but by other aspects of sea acclimatization.Keywords

Thyroid hormones (TH) are major contributor to oxidative stress in mammals because they (1) stimulate reactive oxygen species generation (ROS), (2) impair antioxidant defenses, and (3) increase the susceptibility to free radicals of most tissues. Unlike mammals, THs seem to diminish mitochondrial ROS while they have limited effect on the antioxidant machinery in birds. However, how THs modify the susceptibility to ROS has never been explored in an avian model, and very little is known about their effect on oxidative balance in birds. Therefore, the objective of our study was to examine the effect of chronic pharmacological hypo- and hyperthyroidism on (i) the susceptibility of mitochondrial membranes to ROS; and (ii) the level of oxidative stress assessed by measuring oxidative damage to lipids, nucleic acids and proteins in the gastrocnemius muscle of ducklings. We show that hypothyroidism had no effect on the susceptibility of mitochondrial membranes to free radicals. Hypothyroid ducklings had lower oxidized lipids (−31%) and DNA (−25%) but a similar level of protein carbonylation relative to controls. Conversely, mitochondrial membranes of hyperthyroid ducklings exhibited higher unsaturation (+12%) and peroxidation (+31%) indexes than in controls indicating a greater susceptibility to free radicals. However, hyperthyroid ducklings exhibited more oxidative damages on proteins (+67%) only, whereas lipid damages remained unchanged, and there was a slight reduction (−15%) in damages to DNA compared to euthyroid controls. Our results indicate that birds and mammals present fundamental differences in their oxidative stress response to thyroid status. J. Exp. Zool. 321A: 415–421, 2014. © 2014 Wiley Periodicals, Inc.

Massive bubble formation after diving can lead to decompression sickness (DCS) that can result in neurological disorders. In experimental dives using hydrogen as the diluent gas, decreasing the body's H2burden by inoculating hydrogen-metabolizing microbes into the gut reduces the risk of DCS. In contrast, we have shown that gut bacterial fermentation in rats on a standard diet promotes DCS through endogenous hydrogen production. Therefore, we set out to test these experimental results in humans. Thirty-nine divers admitted into our hyperbaric center with neurological DCS (Affected Divers) were compared with 39 healthy divers (Unaffected Divers). Their last meal time and composition were recorded. Gut fermentation rate was estimated by measuring breath hydrogen 1-4 h after the dive. Breath hydrogen concentrations were significantly higher in Affected Divers (15 ppm [6–23] vs. 7 ppm [3–12]; P = 0.0078). With the use of a threshold value of 16.5 ppm, specificity was 87% [95% confidence interval (CI) 73–95] for association with neurological DCS onset. We observed a strong association between hydrogen values above this threshold and an accident occurrence (odds ratio = 5.3, 95% CI 1.8–15.7, P = 0.0025). However, high fermentation potential foodstuffs consumption was not different between Affected and Unaffected Divers. Gut fermentation rate at dive time seemed to be higher in Affected Divers. Hydrogen generated by fermentation diffuses throughout the body and could increase DCS risk. Prevention could be helped by excluding divers who are showing a high fermentation rate, by eliminating gas produced in gut, or even by modifying intestinal microbiota to reduce fermentation rate during a dive.

Massive bubble formation after diving can lead to decompression sickness (DCS). During dives with hydrogen as a diluent for oxygen, decreasing the body’s H2 burden by inoculating hydrogen-metabolizing microbes into the gut reduces the risk of DCS. So we set out to investigate if colonic fermentation leading to endogenous hydrogen production promotes DCS in fasting rats. Four hours before an experimental dive, 93 fasting rats were force-fed, half of them with mannitol and the other half with water. Exhaled hydrogen was measured before and after force-feeding. Following the hyperbaric exposure, we looked for signs of DCS. A higher incidence of DCS was found in rats force-fed with mannitol than in those force-fed with water (80%, [95%CI 56, 94] versus 40%, [95%CI 19, 64], p

International audience; Repeated deep dives are highly pro-oxidative events for air-breathing aquatic foragers such as penguins. At ␣edging, the transition from a strictly terrestrial to a marine lifestyle may therefore trigger a complex set of anti-oxidant responses to prevent chronic oxidative stress in immature penguins but these pro- cesses are still unde␣ned. By combining in vivo and in vitro approaches with transcriptome analysis, we investigated the adaptive responses of sea-acclimatized (SA) immature king penguins (Aptenodytes pa- tagonicus) compared with pre-␣edging never-immersed (NI) birds. In vivo, experimental immersion into cold water stimulated a higher thermogenic response in SA penguins than in NI birds, but both groups exhibited hypothermia, a condition favouring oxidative stress. In vitro, the pectoralis muscles of SA birds displayed increased oxidative capacity and mitochondrial protein abundance but unchanged reactive oxygen species (ROS) generation per g tissue because ROS production per mitochondria was reduced. The genes encoding oxidant-generating proteins were down-regulated in SA birds while mRNA abundance and activity of the main antioxidant enzymes were up-regulated. Genes encoding proteins involved in repair mechanisms of oxidized DNA or proteins and in degradation processes were also up-regulated in SA birds. Sea life also increased the degree of fatty acid unsaturation in muscle mitochondrial membranes resulting in higher intrinsic susceptibility to ROS. Oxidative damages to protein or DNA were reduced in SA birds. Repeated experimental immersions of NI penguins in cold-water partially mimicked the effects of acclimatization to marine life, modi␣ed the expression of fewer genes related to oxidative stress but in a similar way as in SA birds and increased oxidative damages to DNA. It is concluded that the multi- faceted plasticity observed after marine life may be crucial to maintain redox homeostasis in active tissues subjected to high pro-oxidative pressure in diving birds. Initial immersions in cold-water may initiate an hormetic response triggering essential changes in the adaptive antioxidant response to marine life.

International audience; The passage from shore to marine life is a critical step in the development of juvenile penguins and is characterized by a fuel selection towards lipid oxida- tion concomitant to an enhancement of lipid-induced ther- mogenesis. However, mechanisms of such thermogenic improvement at fledging remain undefined. We used two different groups of pre-fledging king penguins (Apteno- dytes patagonicus) to investigate the specific contribution of cold exposure during water immersion to lipid metabo- lism. Terrestrial penguins that had never been immersed in cold water were compared with experimentally cold-water immersed juveniles. Experimentally immersed penguins underwent ten successive immersions at approximately 9–10 °C for 5 h over 3 weeks. We evaluated adaptive ther- mogenesis by measuring body temperature, metabolic rate and shivering activity in fully immersed penguins exposed to water temperatures ranging from 12 to 29 °C. Both never-immersed and experimentally immersed penguins were able to maintain their homeothermy in cold water, exhibiting similar thermogenic activity. In vivo, perfusion of lipid emulsion at thermoneutrality induced a twofold larger calorigenic response in experimentally immersed than in never-immersed birds. In vitro, the respiratory rates and the oxidative phosphorylation efficiency of isolated muscle mitochondria were not improved with cold-water immersions. The present study shows that acclimation to cold water only partially reproduced the fuel selection towards lipid oxidation that characterizes penguin acclima- tization to marine life.

An overwhelming nitric oxide (NO) production is a crucial step in the circulatory events as well as in the cellular alterations taking place in septic shock. However, evidences of this role arise from studies assessing the NO production on an intermittent basis precluding any clear evaluation of temporal relationship between NO production and circulatory alterations. We evaluated this relationship by using a NO specific electrode allowing a continuous measurement of NO production. Septic shock was induced by a cecal ligation and puncture (CLP) in a first group of anesthetized rats. After the same CLP, a second group received a selective iNOS inhibitor (L-NIL). Control rats were sham operated or sham operated with L-NIL administration. While NO concentration was measured every 2 min by a NO-sensitive electrode over 7h following CLP, the liver microcirculation was recorded by a laser-Doppler flowmeter. CLP induced a severe septic shock with hypotension occurring at a mean time of 240 min after CLP. At the same time, an increase in liver NO concentration was observed, whereas a decrease in microvascular liver perfusion was noted. In the septic shock group, L-NIL administration induced an increase in arterial pressure whereas the liver NO concentration returned to baseline values. In addition, shock groups experienced an increase in iNOS mRNA. These data showed a close temporal relationship between the increase in liver NO concentration and the microvascular alteration taking place in the early period of septic shock induced by CLP. The iNOS isoform is involved in this NO increase.

At hatching, breaking eggshell induces a surge in oxygen availability that is likely to generate oxidative stress in newborn chicks. To investigate the involvement of potential adaptive antioxidant mechanisms, we explored some markers of oxidative stress and the regulation of muscle avian uncoupling protein (avUCP) and adenine nucleotide translocase (ANT) in ducklings in the peri-hatching period. When compared with pre-hatching levels, the amount of peroxidized lipids were increased 24 h after external pipping in gastrocnemius muscle (+37%) and heart (+39%) as well as the muscle avUCP mRNA expression (+60%) but the susceptibility of red blood cells to free radicals (a functional test of oxidative status) was not affected. In order to relate these changes to the oxidative transition of hatching, an imposed hypoxia/re-oxygenation protocol was used. Hatched chicks that had spent the last 24 h of incubation in artificial severe hypoxia showed a rise in muscle (+50%) and heart (+69%) lipid peroxidation, an increased susceptibility of red blood cells to free radicals, a marked over-expression of avUCP mRNA (+105%) and a rise in mitochondrial ANT content (+54%). These results suggest that avian UCP and ANT may contribute to prepare incubating eggs to the oxidative stress generated by the hypoxia/re-oxygenation transition naturally occurring at hatching.

SUMMARY Here we report the partial nucleotide sequence of a reptilian uncoupling protein (repUCP) gene from the European common lizard (Lacerta vivipara). Overlapping sequence analysis reveals that the protein shows 55%, 72% and 77% sequence homology with rat UCP1, UCP2 and UCP3, respectively,and 73% with bird and fish UCPs. RepUCP gene expression was ubiquitously detected in 4°C cold-acclimated lizard tissues and upregulated in muscle tissues by a 20 h exposure to sub-zero temperatures in a supercooling state or after thawing. In parallel, we show an increase in the co-activators,peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α) and peroxisome proliferator-activated receptors (PPAR), mRNA expression, suggesting that the mechanisms regulating UCP expression may be conserved between mammals (endotherms) and reptiles (ectotherms). Furthermore,mitochondria extracted from lizard skeletal muscle showed a guanosine diphosphate (GDP)-sensitive non phosphorylating respiration. This last result indicates an inhibition of extra proton leakage mediated by an uncoupling protein, providing arguments that repUCP is functional in lizard tissues. This result is associated with a remarkable GDP-dependent increase in mitochondrial endogenous H2O2 production. All together, these data support a physiological role of the repUCP in superoxide limitation by lizard mitochondria in situations of stressful oxidative reperfusion following a re-warming period in winter.

6 PAGES; International audience; We describe a method that allows prediction of resting metabolic rate (RMR, ml O2 Æ min1) in adult male and female king penguins on shore by measuring body mass (Mb) and the length of the foot, flipper and beak. This method is accurate, underestimating measured RMR (n=114) by 4% in a data set consisting of 44 birds (33 males and 11 females). Measurement error was unbiased with respect to fasting duration and can therefore estimate RMR during any stage of fasting. This new method provides significant cost and logistical savings when estimating RMR during fieldwork, allowing RMR of a large number of birds to be measured quickly. These findings suggest the possibility that the use of Mb and morphometrics will allow development of general and specific equations to estimate RMR in other species.

King penguins ( Aptenodytes patagonicus) may fast for up to 30 days during their breeding period. As such extended fasting may affect the relationship between the rate of O2consumption (V̇o2) and heart rate ( fH), five male king penguins were exercised at various speeds on repeated occasions during a fasting period of 24–31 days. In addition, V̇o2and fHwere measured in the same animals during rest in cold air and water (4°C). V̇o2and fHat rest and V̇o2during exercise decreased with fasting. There was a significant relation between V̇o2and fH( r2= 0.56) that was improved by including speed, body mass ( Mb), number of days fasting ( t), and a cross term between fHand t ( r2= 0.92). It was concluded that there was a significant change in the V̇o2- fHrelationship with fasting during exercise. As t is measurable in the field and was shown to be significant and, therefore, a practical covariate, a regression equation for use when birds are ashore was obtained by removing speed and Mb. When this equation was used, predicted V̇o2was in good agreement with the observed data, with an overall error of 3.0%. There was no change in the V̇o2- fHrelationship in penguins at rest in water.

Juvenile king penguins develop adaptive thermogenesis after repeated immersion in cold water. However, the mechanisms of such metabolic adaptation in birds are unknown, as they lack brown adipose tissue and uncoupling protein-1 (UCP1), which mediate adaptive non-shivering thermogenesis in mammals. We used three different groups of juvenile king penguins to investigate the mitochondrial basis of avian adaptive thermogenesis in vitro. Skeletal muscle mitochondria isolated from penguins that had never been immersed in cold water showed no superoxide-stimulated proton conductance, indicating no functional avian UCP. Skeletal muscle mitochondria from penguins that had been either experimentally immersed or naturally adapted to cold water did possess functional avian UCP, demonstrated by a superoxide-stimulated, GDP-inhibitable proton conductance across their inner membrane. This was associated with a markedly greater abundance of avian UCP mRNA. In the presence (but not the absence) of fatty acids, these mitochondria also showed a greater adenine nucleotide translocase-catalysed proton conductance than those from never-immersed penguins. This was due to an increase in the amount of adenine nucleotide translocase. Therefore, adaptive thermogenesis in juvenile king penguins is linked to two separate mechanisms of uncoupling of oxidative phosphorylation in skeletal muscle mitochondria: increased proton transport activity of avian UCP (dependent on superoxide and inhibited by GDP) and increased proton transport activity of the adenine nucleotide translocase (dependent on fatty acids and inhibited by carboxyatractylate).

We present the partial nucleotide sequence of the avian uncoupling protein (avUCP) gene from king penguin (Aptenodytes patagonicus), showing that the protein is 88-92% identical to chicken (Gallus gallus), turkey (Meleagris gallopavo), and hummingbird (Eupetomena macroura). We show that superoxide activates the proton conductance of mitochondria isolated from king penguin skeletal muscle. GDP abolishes the superoxide-activated proton conductance, indicating that it is mediated via avUCP. In the absence of superoxide there is no GDP-sensitive component of the proton conductance from penguin muscle mitochondria demonstrating that avUCP plays no role in the basal proton leak.

The rapid maturation of thermoregulatory mechanisms may be of critical importance for optimising chick growth and survival and parental energy investment under harsh climatic conditions. The ontogeny of thermoregulatory mechanisms was studied in growing king penguin chicks from hatching to the full emancipation observed at 1 month of age in the sub-Antarctic area (Crozet Archipelago). Newly hatched chicks showed small, but significant regulatory thermogenesis (21% rise in heat production assessed by indirect calorimetry), but rapidly became hypothermic. Within a few days, both resting (+32%) and peak (+52%) metabolic rates increased. The first week of life was characterised by a two-fold rise in thermogenic capacity in the cold, while thermal insulation was not improved. During the second and third weeks of age, thermal insulation markedly rose (two-fold drop in thermal conductance) in relation to down growth, while resting heat production was slightly reduced (-13%). Shivering (assessed by electromyography) was visible right after hatching, although its efficiency was limited. Thermogenic efficiency of shivering increased five-fold with age during the first weeks of life, but there was no sign of non-shivering thermogenesis. We conclude that thermal emancipation of king penguin chicks may be primarily determined by improvement of thermal insulation after thermogenic processes have become sufficiently matured. Both insulative and metabolic adaptations are required for the rapid ontogeny of thermoregulation and thermal emancipation in growing king penguin chicks.

The control of hepatic metabolism by substrates and hormones was assessed in perfused liver from young Muscovy ducklings. Studies were performed in fed or 24-h fasted 5-week-old thermoneutral (25 degrees C; TN) or cold-acclimated ducklings (4 degrees C; CA) and results were compared with those obtained in rats. Basal oxygen uptake of perfused liver (LVO2) was higher after cold acclimation both in fed (+65%) and 24-h fasted (+29%) ducklings and in 24-h fasted rats (+34%). Lactate (2 mM), the main gluconeogenic substrate in birds, similarly increased LVO2 in both TN and CA ducklings and the effect was larger after fasting. Both glucagon and norepinephrine dose-dependently increased LVO2 in ducklings and rats, but cold acclimation did not improve liver response and liver sensitivity to norepinephrine in ducklings was even reduced in CA animals. Liver contribution to glucagon-induced thermogenesis in vivo was estimated to be 22% in TN and 12% in CA ducklings. Glucagon stimulated gluconeogenesis from lactate in duckling liver and the stimulation was 2.2-fold higher in CA than in TN fasted birds. These results indicate a stimulated hepatic oxidative metabolism in CA ducklings but hepatic glucagon-induced thermogenesis (as measured by LVO2) was not improved. A role of the liver is suggested in duckling metabolic acclimation to cold through an enhanced hepatic gluconeogenesis under glucagon control.

The phospholipid fatty acid (FA) composition and functional properties of skeletal muscle and liver mitochondria were examined in cold-acclimated (CA, 4 degrees C) ducklings. Phospholipid FA of isolated muscle mitochondria from CA birds were longer and more unsaturated than those from thermoneutral (TN, 25 degrees C) reared ducklings. The rise in long-chain and polyunsaturated FA (PUFA, mainly 20:4n-6) was associated with a higher State 4 respiration rate and a lower respiratory control ratio (RCR). Hepatic mitochondria, by contrast, were much less affected by cold acclimation. The cold-induced changes in phospholipid FA profile and functional properties of muscle mitochondria were reproduced by giving TN ducklings a diet enriched in grapeseed oil (GO, rich in n-6 FA), suggesting a causal relationship between the membrane structure and mitochondrial functional parameters. However, hepatic mitochondria from ducklings fed the GO diet also showed an enrichment in long-chain PUFA but opposite changes in their biochemical characteristics (lower State 4, higher RCR). It is suggested that the differential modulation of mitochondrial functional properties by membrane lipid composition between skeletal muscle and liver may depend on muscle-specific factors possibly interacting with long-chain PUFA and affecting the proton leakiness of mitochondrial membranes.

Brown adipose tissue (BAT) is implicated in both cold-induced thermogenesis and regulation of energy expenditure and is mainly controlled by sympathetic innervation. To clarify the permissive and/or complementary roles of glucagon in cold-induced BAT activation, glucagon receptor gene expression and its modulation by sympathetic activity were investigated in rats. One pad of interscapular BAT was surgically denervated while the other pad was sham operated, then rats were either cold-exposed (CE) for 1 week at 4°C or kept near thermoneutrality (25°C, TN). Using a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay, it was shown that cold exposure decreased (-44%) the relative abundance of BAT glucagon receptor mRNA, an effect which was prevented by unilateral surgical sympathectomy of BAT. The present results show a negative control by sympathetic nervous activity of glucagon receptor gene expression and/or of glucagon receptor mRNA stability in BAT of cold-exposed rats. The down-regulation of glucagon receptor expression during cold exposure does not support a major role of the peptide in the thermogenic control of BAT.

The effect of long-term hindlimb unloading (2 or 5 week) on the expression of uncoupling protein-3 (UCP3) gene was investigated in rat skeletal muscles. The interaction of hindlimb unloading and thyroid status was also investigated at 2 weeks. Whatever the duration, mechanical unloading induced a similar increase in UCP3 mRNA relative abundance in the slow-twitch soleus (SOL) muscle (+80%, P < 0.05), whereas no effect was observed in the fast-twitch extensor digitorum longus (EDL) muscle. Hypothyroidism down-regulated while hyperthyroidism up-regulated UCP3 mRNA relative abundance in both SOL and EDL muscles, but thyroid status did not prevent the up-regulation of UCP3 induced by 2 weeks of suspension. These data therefore indicate for the first time that long-term hindlimb unloading up-regulates muscle UCP3 gene expression in a muscle-specific manner which is independent of thyroid status.

To determine whether newborn pigs are able to display adequate cardiovascular adjustments favouring shivering thermogenesis in skeletal muscles soon after birth, regional blood flow and fractional distribution of cardiac output were determined in 1-day-old (n = 6) and 5-day-old (n = 6) conscious piglets at thermal neutrality and during cold exposure, using coloured microspheres. Five-day-old piglets stayed with the sow before the experiment. The cold challenge was designed to induce a similar increase (~+90%) in heat production at both ages. Skeletal muscle blood flow increased with both age (p < 0.05) and cold exposure (p < 0.001), with the effect of cold being more pronounced in 5-day-old piglets than in 1-day-old piglets (+60%, p < 0.05). The difference between individual muscles increased with age, with fractional blood flow being 41% higher in rhomboideus than in longissimus thoracis muscle during cold exposure in 5-day-old piglets (p < 0.05). Cardiac output was similar at both ages and increased by 23% in the cold (p < 0.001). At 1 day of age, there was no redistribution of cardiac output among the internal organs during the cold challenge, while at 5 days of age, the increase in muscle fractional blood flow was associated with a reduction (p < 0.05) in the fraction of cardiac output reaching the skin (-24%), the small intestine (-21%), and the liver (-20%). In conclusion, these results suggest that there is a rapid postnatal improvement of cardiovascular adjustments favouring blood perfusion and probably heat production during cold-induced shivering in the most oxidative muscles studied. This cardiovascular response may play a role in the postnatal enhancement of thermoregulation in piglets.Key words: skeletal muscle, blood distribution, shivering thermogenesis, piglet, age.

Energetic adaptation to fasting in the cold has been investigated in a nocturnal raptor, the barn owl (Tyto alba), during winter. Metabolic rate and body temperature (Tb) were monitored in captive birds, (1) after acute exposure to different ambient temperatures (Ta), and (2) during a prolonged fast in the cold (4 degrees C), to take into account the three characteristic phases of body fuel utilization that occur during a long-term but reversible fast. In postabsorptive birds, metabolic rate in the thermoneutral zone was 4. 1+/-0.1 W kg-1 and increased linearly below a lower critical temperature of 23 degrees C. Metabolic rate was 70% above basal at +4 degrees C Ta. Wet thermal conductance was 0.22 W kg-1 degrees C-1. During fasting in the cold, the mass-specific resting metabolic rate decreased by 16% during the first day (phase I) and remained constant thereafter. The amplitude of the daily rhythm in Tb was only moderately increased during phase II, with a slight lowering (0. 6 degrees C) in minimal diurnal Tb, but rose markedly in phase III with a larger drop (1.4 degrees C) in minimal diurnal Tb. Refeeding the birds ended phase III and reversed the observed changes. These results indicate that diurnal hypothermia may be used in long-term fasting barn owls and could be triggered by a threshold of body lipid depletion, according to the shift from lipid to protein fuel metabolism occurring at the phase II/phase III transition. The high cost of regulatory thermogenesis and the limited use of hypothermia during fasting may contribute to the high mortality of barn owls during winter.

Gastrocnemius subsarcolemmal and intermyofibrillar mitochondria were isolated from 5-week-old cold-acclimated and thermoneutral control ducklings. In vitro respiration (polarography) and ATP synthesis (bioluminescence) were determined at 25°C. Subsarcolemmal mitochondria showed a higher state 4 respiration and lower respiratory control and ADP/O ratio in cold-acclimated than in thermoneutral ducklings. Palmitate decreased the rate of ATP synthesis in both mitochondrial populations to about 30% of maximal but failed to abolish this process even at high concentrations. It is suggested that both expensive ATP synthesis and increased ATP hydrolysis could contribute synergistically to muscle non-shivering thermogenesis in cold-acclimated ducklings.

The influence of thyroid status on glucagon receptor mRNA levels was investigated in rats using a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Glucagon receptor mRNA was detected in liver, brown and white adipose tissues (BAT and WAT) and brain. In BAT and WAT, pharmacologically-induced moderate hypothyroidism resulted in a marked reduction in the relative abundance of glucagon receptor mRNA. Short-term treatment of hypothyroid rats with exogenous 3,3′,5′-triiodo- l -thyronine (T3), resulting in a marked hyperthyroidism, reversed the phenomenon in BAT while the reversal was only partial in WAT. In the liver, there was no significant effect of mild hypothyroidism while there was a positive effect of hyperthyroidism. In brain, the relative tissue abundance of glucagon receptor mRNA was not affected by the large changes in plasma T3. The present results therefore indicate that thyroid status may modulate the relative abundance of glucagon receptor mRNA in a tissue-specific manner.

In cold-acclimated (CA) birds, lipids play a crucial role in regulatory thermogenesis by acting both as substrates for and activators of thermogenic processes. The capacity to supply lipids to thermogenic tissues, which could limit cold thermogenesis, was assessed in CA ducklings (5 wk old, 4°C) and compared with thermoneutral controls (TN, 25°C). In CA ducklings, basal lipolytic activity of adipose tissue fragments was higher (202 ± 9 vs. 130 ± 14 nmol glycerol released ⋅ 100 mg tissue−1⋅ h−1, +55%) than in TN controls, while glucagon had a much higher stimulatory effect (+140 to +500% depending on dose). This was consistent with increased plasma levels of nonesterified fatty acids (FA, +57%) and glycerol (+31%) in vivo. In vitro endothelial lipase activity per organ was higher in CA than in TN ducklings in red gastrocnemius muscle (6.3 ± 0.6 vs. 3.5 ± 0.3 μeq nonesterified FA released per hour, +80%) and liver (+55%). The intracellular FA-binding capacity of (12–18 kDa) proteins was higher in gastrocnemius muscle (+43%) and liver (+74%) from CA ducklings. In gastrocnemius, it was linked to a higher content (21 ± 2 vs. 15 ± 2 μg/mg protein, +37%) of an intracellular 15.4-kDa FA-binding protein. These in vitro results indicate that coordinated increases in FA supply from adipose tissue, cellular uptake of lipoprotein-derived FA, and intracellular FA transport capacity occur in CA ducklings endowed with higher thermogenic capacity and cold endurance.

The humoral modulation of resting muscle heat production of chickens (Gallus domesticus) was investigated in vitro. The resting distal lower limb was perfused via the popliteal artery at 25 degrees C without erythrocytes at constant flow. The preparation was stable for at least 3 hr, showing a constant oxygen uptake (MO2) and perfusion pressure as well as adequately maintaining muscle energy charge and creatine phosphate: creatine ratio. Noradrenaline (NOR), adrenaline (ADR) and serotonin (5-HT) each caused a dose-dependent rise in perfusion pressure. NOR and ADR evoked increased MO2 at low doses eventually followed by decreased MO2 at higher agonist concentrations. 5-HT gave smaller but qualitatively similar MO2 effects. The actions of 50 nM NOR were blocked by prazosin (10 microM) and nitroprusside (0.5 mM), but not altered by propranolol (10 microM). NOR-induced stimulatory MO2 changes in the presence of pharmacological concentrations (1 microM) of glucagon were more pronounced and the thermogenic concentration range of NOR was increased. Taken together, these in vitro findings demonstrate a potential for vasoconstrictor-controlled muscle nonshivering thermogenesis in birds as in marsupials and mammals, suggesting that vascular control of muscle MO2 may be a widespread biological mechanism. The possible implications of these findings for avian nonshivering thermogenesis are discussed.

The possible involvement of locally produced insulin-like growth factor I (IGF-I) in the cold-induced hyperplasia of interscapular brown adipose tissue (BAT) was investigated in 2-, 4-, and 7-day cold-exposed (CE, 4 degrees C) rats by measuring BAT IGF-I expression at a time when extensive BAT cell proliferation occurs. By comparison with thermoneutral (25 degrees C) controls, plasma IGF-I decreased in CE rats despite an increased food intake, whereas BAT IGF-I peptide increased markedly to peak after 4 days at 4 degrees C. The ratio of class 1 to class 2 IGF-I mRNA was much higher in BAT than in liver. BAT IGF-I mRNA levels per unit weight total RNA doubled after 2 days at 4 degrees C but decreased thereafter to the level in controls. Upregulation of BAT IGF-I mRNA also occurred in CE rats with a food intake restricted to the level of controls. The transient cold-induced upregulation of BAT IGF-I (per unit weight total RNA) suggests that IGF-I plays a role in the early cold-induced BAT hyperplasia that occurs in vivo.

Thyroid hormones (TH) are major contributor to oxidative stress in mammals because they (1) stimulate reactive oxygen species generation (ROS), (2) impair antioxidant defenses, and (3) increase the susceptibility to free radicals of most tissues. Unlike mammals, THs seem to diminish mitochondrial ROS while they have limited effect on the antioxidant machinery in birds. However, how THs modify the susceptibility to ROS has never been explored in an avian model, and very little is known about their effect on oxidative balance in birds. Therefore, the objective of our study was to examine the effect of chronic pharmacological hypo- and hyperthyroidism on (i) the susceptibility of mitochondrial membranes to ROS; and (ii) the level of oxidative stress assessed by measuring oxidative damage to lipids, nucleic acids and proteins in the gastrocnemius muscle of ducklings. We show that hypothyroidism had no effect on the susceptibility of mitochondrial membranes to free radicals. Hypothyroid ducklings had lower oxidized lipids (-31%) and DNA (-25%) but a similar level of protein carbonylation relative to controls. Conversely, mitochondrial membranes of hyperthyroid ducklings exhibited higher unsaturation (+12%) and peroxidation (+31%) indexes than in controls indicating a greater susceptibility to free radicals. However, hyperthyroid ducklings exhibited more oxidative damages on proteins (+67%) only, whereas lipid damages remained unchanged, and there was a slight reduction (-15%) in damages to DNA compared to euthyroid controls. Our results indicate that birds and mammals present fundamental differences in their oxidative stress response to thyroid status.

International audience; The evolutionary trade-off between tissue growth and mature function restricts the post natal development of polar birds. The present study uses an original integrative approach as it includes gene expression, plus biochemical and physiological analysis to investigate how Adélie penguin chicks achieve a rapid growth despite the energetic constraints linked to the cold and the very short breeding season in Antarctica. In pectoralis muscle, the main thermogenic tissue in birds, our data show that the transition from ectothermy to endothermy on Day 15 post- hatching is associated with substantial and coordinated changes in the transcription of key genes. While the early activation of genes controlling cell growth and differentiation (avGHR, avIGF-1R, T3Rβ) is rapidly down-regulated after hatching, the global increase in the relative expression of genes involved in thermoregulation (avUCP, avANT, avLPL) and transcriptional regulation (avPGC1α, avT3Rβ) underlie the muscular acquisition of oxidative metabolism. Adélie chicks only become real endotherms at 15 days of age with the development of an oxidative muscle phenotype and the ability to shiver efficiently. The persistent muscular expression of IGF-1 throughout growth probably acts as a local mediator to adjust muscle size and its oxidative capacity to anticipate the new physiological demands of future Dives in cold water. The up-regulation of T3Rβ mRNA levels suggests that circulating T3 may play an important role in the late maturation of skeletal muscle by reinforcing, at least in part, the paracrine action of IGF-1. From day 30, the metabolic shift from mixed substrate to lipid metabolism, with the markedly increased mRNA levels of muscle avLPL, avANT and avUCP, suggests the late development of a fatty acid-enhanced muscle non-shivering thermogenesis mechanism. This molecular control is the key to this finely-tuned strategy by which the Adélie penguin chick successfully heads for the sea on schedule.

International audience; The ontogeny of pectoralis muscle bioenergetics was studied in growing Adelie penguin chicks during the first month after hatching and compared with adults using permeabilized fibers and isolated mitochondria. With pyruvatemalate-succinate or palmitoyl-carnitine as substrates, permeabilized fiber respiration markedly increased during chick growth (3-fold) and further rose in adults (1.4-fold). Several markers of muscle fiber oxidative activity (cytochrome oxidase, citrate synthase, hydroxylacyl-CoA dehydrogenase) increased 6- to 19-fold with age together with large rises in intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondrial content (3- to 5-fold) and oxidative activities (1.5- to 2.4-fold). The proportion of IMF relative to SS mitochondria increased with chick age but markedly dropped in adults. Differences in oxidative activity between mitochondrial fractions were reduced in adults compared with hatched chicks. Extrapolation of mitochondrial to muscle respirations revealed similar figures with isolated mitochondria and permeabilized fibers with carbohydrate-derived but not with lipid-derived substrates, suggesting diffusion limitations of lipid substrates with permeabilized fibers. Two immunoreactive fusion proteins, mitofusin 2 (Mfn2) and optic atrophy 1 (OPA1), were detected by Western blots on mitochondrial extracts and their relative abundance increased with age. Muscle fiber respiration was positively related with Mfn2 and OPA1 relative abundance. Present data showed by two complementary techniques large ontogenic increases in muscle oxidative activity that may enable birds to face thermal emancipation and growth in childhood and marine life in adulthood. The concomitant rise in mitochondrial fusion protein abundance suggests a role of mitochondrial networks in the skeletal muscle processes of bioenergetics that enable penguins to overcome harsh environmental constraints.

International audience; 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 T(3) 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 T(3). 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.

Energy intake profoundly influences many endocrine axes which in turn play a central role in development. The specific influence of a short period of mild hypothyroidism, similar to that induced by undernutrition, in regulating muscle development has been assessed in a large mammal during early postnatal life. Hypothyroidism was induced by providing methimazole and iopanoic acid in the feed of piglets between 4 and 14 d of age, and controls were pair-fed to the energy intake of their hypothyroid littermates. Thyroid status was evaluated, and myofibre differentiation and cation pump concentrations were then assessed in the following functionally distinct muscles:longissimus dorsi(l. dorsi),soleusandrhomboideus. Reductions in plasma concentrations of thyroxine (T4; 32%,P< O·Ol), triiodothyronine (T3;48%,P< 0·001), free T3, (58%,P< 0·001)and hepatic 5'-monodeiodinase (EC1.11.1.8) activity (74%,P< 0·001) occurred with treatment. Small, although significant, increases in the proportion of type I slow-twitch oxidative fibres occurred with mild hypothyroidism, inl. dorsi(2%,P< 0·01) andsoleus(7%,P< 0·01). Nuclear T3-receptor concentration inl. dorsiof hypothyroid animals compared with controls increased by 46% (P< 0·001), a response that may represent a homeostatic mechanism making muscle more sensitive to low levels of circulating thyroid hormones. Nevertheless, Na+, K+-ATPase (EC3.6.1.37) concentration was reduced by 15–16% in all muscles (l.dorsiP< 0·05,soleusP< 0·001,rhomboideusP< 0·05), and Ca2+-ATPase (EC3.6.1.38) concentration was significantly reduced in the two slow-twitch muscles: by 22% inrhomboideus(P< 0·001) and 23% insoleus(P< 0·05). It is concluded that during early postnatal development of large mammals a period of mild hypothyroidism, comparable with that found during undernutrition, induces changes in myofibre differentiation and a down-regulation of cation pumps in skeletal muscle. Such changes would result in slowness of movement and muscle weakness, and also reduce ATP hydrolysis with a concomitant improvement in energetic efficiency.

A complementary deoxyribonucleic acid (cDNA) clone encoding an alpha thyroid hormone receptor (TR alpha) from muscovy duckling liver was isolated and sequenced. Comparison with the chicken TR alpha sequence showed a high degree of homology. Despite 45 nucleotide substitutions, the deduced peptide sequence was similar. This cDNA was used as a probe to characterize the TR alpha mRNA transcripts expressed in muscovy duckling liver and skeletal muscle.

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.

International audience; Insulin is involved in multiple regulatory mechanisms, including body weight and food intake, and plays a critical role in metabolic disorders such as obesity and diabetes. An increasing body of evidence indicates that insulin is also involved in the modulation of olfactory function. The olfactory bulb (OB) contains the highest level of insulin and insulin receptors (IRs) in the brain. However, a role for insulin in odor detection and sniffing behavior remains to be elucidated. Using a behavioral paradigm based on conditioned olfactory aversion (COA) to isoamyl-acetate odor, we demonstrated that an intracerebroventricular (ICV) injection of 14 mU insulin acutely decreased olfactory detection of fasted rats to the level observed in satiated animals. In addition, whereas fasted animals demonstrated an increase in respiratory frequency upon food odor detection, this effect was absent in fasted animals receiving a 14 mU insulin ICV injection as well as in satiated animals. In parallel, we showed that the OB and plasma insulin levels were increased in satiated rats compared to fasted rats, and that a 14 mU insulin ICV injection elevated the OB insulin level of fasted rats to that of satiated rats. We further quantified insulin receptors (IRs) distribution and showed that IRs are preferentially expressed in the caudal and lateral parts of the main OB, with the highest labeling found in the mitral cells, the main OB projection neurons. Together, these data suggest that insulin acts on the OB network to modulate olfactory processing and demonstrate that olfactory function is under the control of signals involved in energy homeostasis regulation and feeding behaviors.

The passage from shore to marine life of juvenile penguins represents a major energetic challenge to fuel intense and prolonged demands for thermoregulation and locomotion. Some functional changes developed at this crucial step were investigated by comparing pre-fledging king penguins with sea-acclimatized (SA) juveniles (Aptenodytes patagonicus). Transcriptomic analysis of pectoralis muscle biopsies revealed that most genes encoding proteins involved in lipid transport or catabolism were upregulated, while genes involved in carbohydrate metabolism were mostly downregulated in SA birds. Determination of muscle enzymatic activities showed no changes in enzymes involved in the glycolytic pathway, but increased 3-hydroxyacyl-CoA dehydrogenase, an enzyme of the β-oxidation pathway. The respiratory rates of isolated muscle mitochondria were much higher with a substrate arising from lipid metabolism (palmitoyl-l-carnitine) in SA juveniles than in terrestrial controls, while no difference emerged with a substrate arising from carbohydrate metabolism (pyruvate).In vivo, perfusion of a lipid emulsion induced a fourfold larger thermogenic effect in SA than in control juveniles. The present integrative study shows that fuel selection towards lipid oxidation characterizes penguin acclimatization to marine life. Such acclimatization may involve thyroid hormones through their nuclear beta receptor and nuclear coactivators.

It has been demonstrated previously that the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) concentration (total concentration of [3H]ouabain-binding sites = maximal [3H]ouabain-binding capacity, Bmax) in longissimus dorsi muscle is higher in young pigs living in a cold than in a warm environment, with the effect being even greater in those on a low rather than a high energy intake in the cold. The importance of thyroid status, myofiber type and size, and muscular activity in determining the concentration of Na(+)-K(+)-ATPase has therefore been assessed in longissimus dorsi, semitendinosus, and soleus muscles from 8-wk-old littermate pigs, which had been living 35 or 10 degrees C on either a high or a low energy intake for 3-4 wk. Changes in neither plasma thyroid hormone concentrations nor myofiber type could account for the greater Bmax at 10 vs. 35 degrees C observed in all three muscles; instead, it was possibly related to the greater muscular activity associated with shivering in the cold. More than 50% of the increase in Bmax on the low compared with the high intake in the cold could be attributed to the smaller fiber size and hence greater membrane area in animals on the low intake, and it is concluded that membrane surface area must be assessed in studies of membrane-bound enzymes.