Claude Duchamp

An uncoupling protein homologue putatively involved in facultative muscle thermogenesis in birds

The cDNA of an uncoupling protein (UCP) homologue was obtained by screening a chicken skeletal-muscle library. The predicted 307-amino-acid sequence of avian UCP (avUCP) is 55, 70, 70 and 46% identical with mammalian UCP1, UCP2 and UCP3 and plant UCP respectively. avUCP mRNA expression is restricted to skeletal muscle and its abundance was increased 1.3-fold in a chicken line showing diet-induced thermogenesis, and 3.6- and 2.6-fold in cold-acclimated and glucagon-treated ducklings developing muscle non-shivering thermogenesis respectively. The present data support the implication of avUCP in avian energy expenditure.

Uncoupling protein and ATP/ADP carrier increase mitochondrial proton conductance after cold adaptation of king penguins

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).

Multilocular adipocytes from muscovy ducklings differentiated in response to cold acclimation.

Morphological and functional aspects of adipose tissue from 6‐week‐old cold‐acclimated muscovy ducklings reared at 4 degrees C ambient temperature (Ta) from the age of 1 week were examined for the occurrence of brown adipose tissue (b.a.t.) in order to explain non‐shivering thermogenesis (n.s.t.) observed at this age. Metabolic rate and integrated muscle electrical activity (e.m.g.) were measured at different Ta (from ‐10 to +28 degrees C) in cold‐acclimated and in control ducklings reared at thermoneutrality. The results confirm the existence of n.s.t. in 6‐week‐old cold‐acclimated muscovy ducklings. In cold‐acclimated ducklings, typical multilocular adipocytes were found in subcutaneous adipose deposits instead of the unilocular white adipocytes as in control ducklings. Mitochondria isolated from this differentiated tissue were less abundant than in b.a.t. of mammals. Their respiration rate was similar to the respiration rate of white adipose tissue mitochondria from control rats and much lower than the b.a.t. mitochondria rate from cold‐acclimated rats. It is therefore unlikely that this differentiated adipose tissue contributes to the n.s.t. observed, an n.s.t. whose capacity reached 5.26 W/kg (+73.5% above resting metabolic rate) in cold‐acclimated ducklings. The role of this differentiated adipose tissue in the metabolic adaptation to cold is discussed.

Nutritional regulation of growth hormone receptor gene expression.

The role of energy intake in regulating growth hormone receptor (GHR) gene expression has been assessed in young growing pigs living at thermal neutrality (26°C) for a 4‐wk period. To determine the importance of altering metabolic demand while maintaining food intake constant, littermates were also studied in a cold environment (10°C). Results were tissue‐specific: the level of GHR mRNA per unit total RNA in liver was greater on high than low (high = 2 × low) food intake (P < 0.001), whereas in muscle it was elevated on the low compared with the high intake diet (P < 0.02) and also at 10°C compared with 26°C (P < 0.04). When results for GHR mRNA were expressed per unit weight of tissue, only the effects of diet on liver and temperature on muscle remained significant. The changes in hepatic GHR mRNA may have been driven in part by nutritionally induced changes in thyroid status, because both plasma 3,5,3‘‐triiodothyronine concentration and liver 5‘‐deiodinase activity were greater on the high than the low intake diet (P < 0.001). Levels of liver GHR mRNA probably had a direct influence on growth of the animals, as they were positively correlated with plasma IGF‐I and growth rate (P < 0.001), whereas muscle GHR mRNA may have had a metabolic role when energy supplies were limited.— Dauncey, M. J., Burton, K. A., White, P., Harrison, A. P., Gilmour, R. S., Duchamp, C., Cattaneo, D. Nutritional regulation of growth hormone receptor gene expression. FASEB J. 8: 81‐88; 1994.

Sleep changes in fasting rats

The proportion and the distribution of wakefulness (W) slow-wave sleep (SWS) and paradoxical sleep (PS) were studied in 27-week-old rats over 24 hr periods, both in the fed state and after having been deprived of food for 2 to 3 weeks. In these rodents, prolonged fasting has been characterized by 3 successive metabolic phases which have been found to correspond to changes in protein metabolism. Sleep-waking changes were not studied during the first phase which was often of short duration (24 hr). During the second phase, i.e., when proteins were spared, the 24 hr proportions of W and sleep states remained unchanged. There were, however, profound changes in the daily mean episodic characteristics of each vigilance state (duration and frequency) except in the case of PS. During the phase II, the differences in the day/night proportions observed in each vigilance state were less than in the fed state. This reflected a lowering in the amplitude of their daily rhythms. In contrast, when protein use rose (phase III), W was increased sharply at the expense of SWS and PS, the latter being almost completely suppressed. During this last phase, which was also of short duration (by mean 3 days) alertness was greatly enhanced and the rats, which were typically nocturnal when fed, became diurnal. The changes in sleep and wakefulness were examined in relation to their effects on the homeostatic and cyclic components of sleep mechanisms and adaptive strategy to food deprivation in rat.

Histochemical arguments for muscular non‐shivering thermogenesis in muscovy ducklings.

1. The histochemical characteristics of gastrocnemius muscle were investigated in 6‐week‐old cold‐acclimated (5 weeks, 4 degrees C) and glucagon‐treated (5 weeks, 25 degrees C, 103 nmol/kg I.P. twice daily) muscovy ducklings, two groups able to develop non‐shivering thermogenesis in vivo. A comparison was made with thermoneutral controls (25 degrees C) of the same age. All animals were fed ad libitum. Fibre type, fibre area and capillary supply have been studied. Further, a quantitative histochemical method for mitochondrial Mg(2+)‐ATPase activity was developed to characterize the mitochondrial coupling state in situ. 2. White gastrocnemius was composed of fast glycolytic (FG) and fast oxidative glycolytic (FOG) fibres, while red gastrocnemius contained FOG and slow oxidative (SO) fibres. In white gastrocnemius, the proportion of FG fibres was higher in glucagon‐treated than in control or cold‐acclimated ducklings. In red gastrocnemius, the proportion of SO fibres was higher in both cold‐acclimated and glucagon‐treated ducklings than in controls. The area of all fibres was generally lower in glucagon‐treated than in other ducklings. 3. The capillary density was higher in both red and white components of the gastrocnemius muscle in cold‐acclimated and glucagon‐treated than in control ducklings, as a result of an increased number of capillaries around each fibre. 4. In all fibres, except the FG type in cold‐acclimated ducklings, the staining intensity of the Mg(2+)‐ATPase reaction was higher in cold‐acclimated and glucagon‐treated than in control ducklings whereas the staining intensity with maximal decoupling of oxidative phosphorylation by dinitrophenol was unchanged. This indicated a more loose‐coupled state of mitochondria in situ in all fibres of cold‐acclimated ducklings, and in FOG fibres of white gastrocnemius and SO fibres of red gastrocnemius in glucagon‐treated ducklings. 5. These results indicated a higher oxidative metabolism of skeletal muscle in both cold‐acclimated and glucagon‐treated than in control ducklings, and for most of the parameters studied, a similarity between cold acclimation and glucagon treatment. Because of the higher loose‐coupled state of muscle mitochondria in cold‐acclimated and glucagon‐treated than in control ducklings, the higher oxidative capacity of skeletal muscle in these ducklings could be used for heat production rather than ATP synthesis and account for muscular non‐shivering thermogenesis.

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.

Differential regulation of uncoupling protein-1, -2 and -3 gene expression by sympathetic innervation in brown adipose tissue of thermoneutral or cold-exposed rats.

The control of uncoupling protein‐1, ‐2 and ‐3 (UCP‐1, UCP‐2, UCP‐3) mRNA levels by sympathetic innervation in rats was investigated by specific and sensitive RT‐PCR assays. In rats reared at thermoneutrality (25°C), unilateral surgical sympathetic denervation of interscapular brown adipose tissue (BAT) markedly reduced the UCP‐1 mRNA level (−38%) as compared with the contralateral innervated BAT pad, but was without significant effect on UCP‐2 and ‐3 mRNA levels. Cold exposure (7 days, 4°C) markedly increased UCP‐1 (+180%), UCP‐2 (+115%) and UCP‐3 (+195%) mRNA levels in interscapular BAT. Unilateral sympathetic denervation prevented the cold‐induced rise in BAT UCP‐1 and UCP‐2 mRNAs, but not that in BAT UCP‐3 mRNA. Results were confirmed by Northern blot analysis. These data indicate a differential endocrine control of UCP‐1, UCP‐2 and UCP‐3 gene expression in rat BAT both at thermoneutrality and during prolonged cold exposure.

Up-regulation of avian uncoupling protein in cold-acclimated and hyperthyroid ducklings prevents reactive oxygen species production by skeletal muscle mitochondria

BackgroundAlthough identified in several bird species, the biological role of the avian homolog of mammalian uncoupling proteins (avUCP) remains extensively debated. In the present study, the functional properties of isolated mitochondria were examined in physiological or pharmacological situations that induce large changes in avUCP expression in duckling skeletal muscle.ResultsThe abundance of avUCP mRNA, as detected by RT-PCR in gastrocnemius muscle but not in the liver, was markedly increased by cold acclimation (CA) or pharmacological hyperthyroidism but was down-regulated by hypothyroidism. Activators of UCPs, such as superoxide with low doses of fatty acids, stimulated a GDP-sensitive proton conductance across the inner membrane of muscle mitochondria from CA or hyperthyroid ducklings. The stimulation was much weaker in controls and not observed in hypothyroid ducklings or in any liver mitochondrial preparations. The production of endogenous mitochondrial reactive oxygen species (ROS) was much lower in muscle mitochondria from CA and hyperthyroid ducklings than in the control or hypothyroid groups. The addition of GDP markedly increased the mitochondrial ROS production of CA or hyperthyroid birds up to, or above, the level of control or hypothyroid ducklings. Differences in ROS production among groups could not be attributed to changes in antioxidant enzyme activities (superoxide dismutase or glutathione peroxidase).ConclusionThis work provides the first functional in vitro evidence that avian UCP regulates mitochondrial ROS production in situations of enhanced metabolic activity.

Long-term fasting decreases mitochondrial avian UCP-mediated oxygen consumption in hypometabolic king penguins

In endotherms, regulation of the degree of mitochondrial coupling affects cell metabolic efficiency. Thus it may be a key contributor to minimizing metabolic rate during long periods of fasting. The aim of the present study was to investigate whether variation in mitochondrial avian uncoupling proteins (avUCP), as putative regulators of mitochondrial oxidative phosphorylation, may contribute to the ability of king penguins (Aptenodytes patagonicus) to withstand fasting for several weeks. After 20 days of fasting, king penguins showed a reduced rate of whole animal oxygen consumption (Vo2; -33%) at rest, together with a reduced abundance of avUCP and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC1-alpha) mRNA in pectoralis muscle (-54%, -36%, respectively). These parameters were restored after the birds had been refed for 3 days. Furthermore, in recently fed, but not in fasted penguins, isolated muscle mitochondria showed a guanosine diphosphate-inhibited, fatty acid plus superoxide-activated respiration, indicating the presence of a functional UCP. It was calculated that variation in mitochondrial UCP-dependent respiration in vitro may contribute to nearly 20% of the difference in resting Vo2 between fed or refed penguins and fasted penguins measured in vivo. These results suggest that the lowering of avUCP activity during periods of long-term energetic restriction may contribute to the reduction in metabolic rate and hence the ability of king penguins to face prolonged periods of fasting.