Jean-Louis Rouanet

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.

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.

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.

Increase in the adenine nucleotide translocase content of duckling subsarcolemmal mitochondria during cold acclimation

Intermyofibrillar and subsarcolemmal mitochondria were isolated from duckling gastrocnemius muscle. The adenine nucleotide translocase (ANT) content of subsarcolemmal mitochondria was found to be half of that present in intermyofibrillar mitochondria. In addition, cold acclimation resulted in a 1.7‐fold increase in subsarcolemmal mitochondrial ANT content, with intermyofibrillar mitochondrial ANT remaining constant. This change in mitochondrial ANT content correlates with the previously reported cold‐induced change in the sensitivity of mitochondria to palmitate‐inhibited ATP synthesis [Roussel et al. (1998) FEBS Lett. 439, 258–262]. It is suggested that the mitochondrial ANT content enhances or reduces the fatty acid uncoupling activity in tissue, depending on the energetic state of mitochondria.

Effects of cold acclimation and palmitate on energy coupling in duckling skeletal muscle mitochondria

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.

Ontogeny of thermoregulatory mechanisms in king penguin chicks (Aptenodytes patagonicus).

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.

Increased in vitro fatty acid supply and cellular transport capacities in cold-acclimated ducklings (Cairina moschata).

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.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 degrees C) and compared with thermoneutral controls (TN, 25 degrees 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 microeq 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 microg/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.

Cold acclimation or grapeseed oil feeding affects phospholipid composition and mitochondrial function in duckling skeletal muscle

The phospholipid fatty acid (FA) composition and functional properties of skeletal muscle and liver mitochondria were examined in cold-acclimated (CA, 4°C) ducklings. Phospholipid FA of isolated muscle mitochondria from CA birds were longer and more unsaturated than those from thermoneutral (TN, 25°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.