Loïc Teulier

Cold-acclimation-induced non-shivering thermogenesis in birds is associated with upregulation of avian UCP but not with innate uncoupling or altered ATP efficiency

SUMMARY Despite their lack of brown adipose tissue, some bird species develop regulatory non-shivering thermogenesis (NST) of skeletal muscle origin in response to cold acclimation. Mechanisms involved in avian NST are still unclear but may involve reduced energetic coupling in skeletal muscle mitochondria through the expression of an avian homologue of mammalian uncoupling proteins. The aim of this work was to investigate whether the expression of avian uncoupling protein (avUCP) would correlate with the capacity for cold-induced muscle NST. Various levels of cold acclimation were obtained by rearing 1-week-old ducklings (Cairina moschata) for 4 weeks at three different ambient temperatures (25°C, 11°C or 4°C). Muscle NST was measured by simultaneous recordings of metabolic rate and electromyographic activity (gastrocnemius muscle) at ambient temperatures (Ta) ranging from 27°C to −5°C. The expression of avUCP gene and mitochondrial bioenergetics were also determined in gastrocnemius muscle. Results showed that muscle NST capacity depends on the Ta at which ducklings were acclimated, i.e. the lower the rearing temperature, the higher the capacity for NST. This increased metabolic heat production occurred in parallel with an upregulation of avUCP, which was not associated with a change in mitochondrial membrane conductance. The intensity of mitochondrial oxidative phosphorylation also increased in proportion with the harshness of cold, while the efficiency of ATP generation was equally effective in all three acclimation temperatures. In the absence of mitochondrial uncoupling, these data indicate a clear link between avUCP expression and the capacity of ducklings to adjust their muscular aerobic activity to cold exposure.

Functional argument for the existence of an avian nitric oxide synthase in muscle mitochondria: Effect of cold acclimation

We report the first evidence of a mitochondrial NO synthase (mtNOS) in bird skeletal muscle. In vitro, mtNOS activity stimulated by l‐arginine reduced intermyofibrillar mitochondrial oxygen uptake and ATP synthesis rates, stimulated endogenous H2O2 generation, but had no effect on oxidative phosphorylation efficiency. Arginine‐induced effects were fully reversed by l‐NAME, a known NOS inhibitor. When ducklings were cold exposed for 4 weeks, muscle mitochondria displayed an increased state 3 respiration, a reduced H2O2 generation but no significant alteration in mtNOS activity. We conclude that mtNOS is expressed in avian skeletal muscle.

Lactate kinetics of rainbow trout during graded exercise: do catheters affect the cost of transport?

SUMMARY Changes in lactate kinetics as a function of exercise intensity have never been measured in an ectotherm. Continuous infusion of a tracer is necessary to quantify rates of lactate appearance (Ra) and disposal (Rd), but it requires double catheterization, which could interfere with swimming. Using rainbow trout, our goals were to: (1) determine the potential effects of catheters and blood sampling on metabolic rate (ṀO2), total cost of transport (TCOT), net cost of transport (NCOT) and critical swimming speed (Ucrit), and (2) monitor changes in lactate fluxes during prolonged, steady-state swimming or graded swimming from rest to Ucrit. This athletic species maintains high baseline lactate fluxes of 24 μmol kg−1 min−1 that are only increased at intensities >2.4 body lengths (BL) s−1 or 85% Ucrit. As the fish reaches Ucrit, Ra is more strongly stimulated (+67% to 40.4 μmol kg−1 min−1) than Rd (+41% to 34.7 μmol kg−1 min−1), causing a fourfold increase in blood lactate concentration. Without this stimulation of Rd during intense swimming, lactate accumulation would double. By contrast, steady-state exercise at 1.7 BL s−1 increases lactate fluxes to ~30 μmol kg−1 min−1, with a trivial mismatch between Ra and Rd that only affects blood concentration minimally. Results also show that the catheterizations and blood sampling needed to measure metabolite kinetics in exercising fish have no significant impact on ṀO2 or TCOT. However, these experimental procedures affect locomotion energetics by increasing NCOT at high speeds and by decreasing Ucrit.

Proline as a fuel for insect flight: enhancing carbohydrate oxidation in hymenopterans.

Bees are thought to be strict users of carbohydrates as metabolic fuel for flight. Many insects, however, have the ability to oxidize the amino acid proline at a high rate, which is a unique feature of this group of animals. The presence of proline in the haemolymph of bees and in the nectar of plants led to the hypothesis that plants may produce proline as a metabolic reward for pollinators. We investigated flight muscle metabolism of hymenopteran species using high-resolution respirometry performed on permeabilized muscle fibres. The muscle fibres of the honeybee, Apis mellifera, do not have a detectable capacity to oxidize proline, as those from the migratory locust, Locusta migratoria, used here as an outgroup representative. The closely related bumblebee, Bombus impatiens, can oxidize proline alone and more than doubles its respiratory capacity when proline is combined with carbohydrate-derived substrates. A distant wasp species, Vespula vulgaris, exhibits the same metabolic phenotype as the bumblebee, suggesting that proline oxidation is common in hymenopterans. Using a combination of mitochondrial substrates and inhibitors, we further show that in B. impatiens, proline oxidation provides reducing equivalents and electrons directly to the electron transport system. Together, these findings demonstrate that some bee and wasp species can greatly enhance the oxidation of carbohydrates using proline as fuel for flight.

Selective upregulation of lipid metabolism in skeletal muscle of foraging juvenile king penguins: an integrative study

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.

Ontogeny of non-shivering thermogenesis in Muscovy ducklings (Cairina moschata).

In precocial birds, developing the capacity for early regulatory thermogenesis appears as a fundamental prerequisite for survival and growth in cold environments. However, the exact nature of these processes has not been thoroughly investigated. Several bird species, such as Muscovy ducks (Cairina moschata), develop muscular non-shivering thermogenesis when chronically exposed to cold. The aim of this study was to investigate the age-dependent development of non-shivering thermogenesis in ducklings reared either at thermoneutrality (25°C) or in the cold (4°C). Non-shivering thermogenesis was assessed weekly by simultaneously measuring whole body metabolic heat production and electromyographic activity during shivering at different temperatures ranging from 29°C to 0°C. We found that ducklings reared at thermoneutrality displayed a capacity for non-shivering thermogenesis during the first month of post-hatching life. This thermogenic mechanism increased further in ducklings chronically exposed to a cold environment, but it decreased over time when birds were kept in a thermoneutral environment.

Metabolic response to lipid infusion in fasting winter-acclimatized king penguin chicks (Aptenodytes patagonicus).

During the cold austral winter, king penguin chicks are infrequently fed by their parents and thus experience severe nutritional deprivation under harsh environmental conditions. These energetic constraints lead to a range of energy sparing mechanisms balanced by the maintenance of efficient thermogenic processes. The present work investigated whether the high thermogenic capacities exhibited by winter-acclimatized king penguin chicks could be related to an increase in lipid substrate supply and oxidation in skeletal muscle, the main site of thermogenesis in birds. To test this hypothesis, we examined i) the effect of an experimental rise in plasma triglyceride on the whole metabolic rate in winter-acclimatized (WA) and de-acclimatized king penguin chicks kept at thermoneutrality (TN), and ii) investigated the fuel preference of muscle mitochondria. In vivo, a perfusion of a lipid emulsion induced a small 10% increase of metabolic rate in WA chicks but not in TN group. In vitro, the oxidation rate of muscle mitochondria respiring on lipid-derived substrate was +40% higher in WA chicks than in TN, while no differences were found between groups when mitochondria oxidized carbohydrate-derived substrate or succinate. Despite an enhanced fuel selection towards lipid oxidation in skeletal muscle, a rise of circulating lipids per se was not sufficient to fully unravel the thermogenic capacity of winter-acclimatized king penguin chicks.

Novel energy-saving strategies to multiple stressors in birds: the ultradian regulation of body temperature

This study aimed to examine thermoregulatory responses in birds facing two commonly experienced stressors, cold and fasting. Logging devices allowing long-term and precise access to internal body temperature were placed within the gizzards of ducklings acclimated to cold (CA) (5°C) or thermoneutrality (TN) (25°C). The animals were then examined under three equal 4-day periods: ad libitum feeding, fasting and re-feeding. Through the analysis of daily as well as short-term, or ultradian, variations of body temperature, we showed that while ducklings at TN show only a modest decline in daily thermoregulatory parameters when fasted, they exhibit reduced surface temperatures from key sites of vascular heat exchange during fasting. The CA birds, on the other hand, significantly reduced their short-term variations of body temperature while increasing long-term variability when fasting. This phenomenon would allow the CA birds to reduce the energetic cost of body temperature maintenance under fasting. By analysing ultradian regulation of body temperature, we describe a means by which an endotherm appears to lower thermoregulatory costs in response to the combined stressors of cold and fasting.

Mitochondrial oxidative phosphorylation efficiency is upregulated during fasting in two major oxidative tissues of ducklings

Fasted endothermic vertebrates must develop physiological responses to maximize energy conservation and survival. The aim of this study was to determine the effect of 1-wk. fasting in 5-wk. old ducklings (Cairina moschata) from whole-body resting metabolic rate and body temperature to metabolic phenotype of tissues and mitochondrial coupling efficiency. At the level of whole organism, the mass-specific metabolic rate of ducklings was decreased by 40% after 1-wk. of fasting, which was associated with nocturnal Tb declines and shallow diurnal hypothermia during fasting. At the cellular level, fasting induced a large reduction in liver, gastrocnemius (oxidative) and pectoralis (glycolytic) muscle masses together with a fuel selection towards lipid oxidation and ketone body production in liver and a lower glycolytic phenotype in skeletal muscles. At the level of mitochondria, fasting induced a reduction of oxidative phosphorylation activities and an up-regulation of coupling efficiency (+30% on average) in liver and skeletal muscles. The present integrative study shows that energy conservation in fasted ducklings is mainly achieved by an overall reduction in mitochondrial activity and an increase in mitochondrial coupling efficiency, which would, in association with shallow hypothermia, increase the conservation of endogenous fuel stores during fasting.

Leanness of Lou/C rats does not require higher thermogenic capacity of brown adipose tissue

Lou/C rats, an inbred strain of Wistar origin, remain lean throughout life and therefore represent a remarkable model of obesity resistance. To date, the exact mechanisms responsible for the leanness of Lou/C rats remain unknown. The aim of the present study was to investigate whether the leanness of Lou/C rats relies on increased thermogenic capacities in brown adipose tissue (BAT). Results showed that although daily energy expenditure was higher in Lou/C than in Wistar rats, BAT thermogenic capacity was not enhanced in Lou/C rats kept at thermoneutrality as demonstrated by reduced thermogenic response to norepinephrine in vivo, similar oxidative activity of BAT isolated mitochondria in vitro, similar levels of UCP1 mRNA and lower abundance of UCP1 protein in interscapular BAT depots. Relative abundance of β3-adrenergic receptor mRNA was lower in Lou/C BAT while that of GLUT4, FABP or CPT1 was not altered. Activity-related energy expenditure was however considerably increased at thermoneutrality as Lou/C rats demonstrated an impressively high spontaneous running activity in voluntary running wheels. Prolonged cold-exposure (4 °C) depressed the spontaneous running activity of Lou/C rats while BAT thermogenic capacity was increased as reflected by rises in BAT mass, oxidative activity and UCP1 expression. It is concluded that the leanness of Lou/C rats cannot be ascribed to higher thermogenic capacity of brown fat but rather to, at least in part, increased locomotor activity. BAT is not deficient in this rat strain as it can be stimulated by cold exposure when locomotor activity is reduced suggesting some substitution between these thermogenic processes.