Hervé Barré

COMPARATIVE STUDY ON THE BEHAVIORAL, VENTILATORY, AND RESPIRATORY RESPONSES OF HYPOGEAN AND EPIGEAN CRUSTACEANS TO LONG-TERM STARVATION AND SUBSEQUENT FEEDING

Abstract Survival, oxygen consumption, locomotory activity and ventilatory activity were recorded during a 180-day starvation period and a subsequent 15-day feeding phase in 3 hypogean crustaceans, Niphargus rhenorhodanensis, Niphargus virei, and Stenasellus virei. For comparison, these parameters were also recorded during a 28-day starvation period and a subsequent 7-day feeding phase in two morphologically close epigean crustaceans, Gammarus fossarum and Asellus aquaticus. Hypogean crustaceans were better adapted to lack of food than epigean ones and all crustaceans previously studied, with survival times largely longer than 200 days. During long-term starvation, the locomotory, ventilatory, and respiratory rates were drastically lowered in subterranean species, whereas surface species showed lower decreases in these rates and responded by a marked and transitory hyperactivity. The higher reduction in metabolic rate by hypogean species would ensure their survival during prolonged periods of food deprivation. We propose an energy strategy for food-limited hypogean crustaceans involving the ability 1) to withstand long-term starvation, and 2) to use the consumed food very efficiently. Resistance to starvation would probably involve a state of temporary torpor during which the subterranean crustaceans subsist on a high energy reserve, such as lipid stores.

Increased oxidative capacity in skeletal muscles from cold-acclimated ducklings: a comparison with rats

1. The effects of prolonged cold exposure on cytochrome oxidase activity were investigated in skeletal muscles, liver and adipose tissues from cold-acclimated (CA) and control (TN) ducklings and rats. 2. Cold acclimation increased the oxidative capacity of skeletal muscles (+33% in gastrocnemius and +195% in pectoral) and liver (+47%) from CA ducklings, but decreased the oxidative capacity of gastrocnemius muscle (-22%) from CA rats. On the other hand, in these CA rats it increased the oxidative capacity of liver by 88% and, above all, brown adipose tissue by 544%. 3. The significance of these changes due to acclimation to cold in ducklings and rats is discussed. Such an increase in oxidative capacity of CA duckling muscles may explain the non-shivering thermogenesis observed in these birds.

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.

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.

Metabolic and insulative changes in winter- and summer-acclimatized King Penguin chicks

SummaryThe effect of photoperiod and cold on metabolic and insulative changes was investigated in winter- and summer-acclimatized King Penguin chicks under natural conditions (in subantarctic climate). Non-shivering thermogenesis was also studied.Metabolic rate of winter-fasting huddling chicks was compared to that of experimental summer-fasting chicks. Metabolic rate was estimated from measurements of loss in body mass and from measurements of O2 consumption and CO2 production at different ambient temperatures (from −40 to +40°C). Shivering activity was estimated.1.The resting metabolic rate (RMR) at thermoneutrality (Fig. 5) was lower (−18%) during winter (2.15 W·kg−1) than during summer (2.63 W·kg−1). The lower critical temperature of the winter-chicks was lower (LCT=−10°C) than in summer-chicks (LCT=4.8°C).2.The thermal conductance in summer-acclimatized chicks (1.78 W·m−2·°C−1 at the LCT) is in the range of that of other large birds (Table 1). However, in winter-acclimatized checks it is 48% less, which makes it the lowest (0.93 W·m−2. °C−1) among the large birds.3.Huddling behavior allows the winter-acclimatized chicks to decrease metabolic rate below the RMR and to increase the length of total fasting (84±3 days in winter v.s. 44±3 days in summer).

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.