Effect of cold-acclimation on oxygen uptake and glucose production of perfused duckling liver.

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.

Muscle-specific up-regulation of rat UCP3 mRNA expression by long-term hindlimb unloading.

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.

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 degrees 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 degrees 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.

Tissue-specific modulation of rat glucagon receptor mRNA by thyroid status.

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.

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 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.

Hormonal control of thermogenesis in perfused muscle of Muscovy ducklings.

Endocrine stimulation of muscle nonshivering thermogenesis (NST) in ducklings was investigated in vitro using a perfused hindlimb preparation maintained at 25 degrees C. Effects of flow rate, norepinephrine (NE), epinephrine, and glucagon on perfused muscle oxygen consumption (MO2) and perfusion pressure were studied. Control ducklings (Cairina moschata, 5 wk old) reared at thermoneutrality (25 degrees C, TN) were compared with two age-matched groups exhibiting muscle NST in vivo: cold-acclimated ducklings (4 degrees C, 4 wk, CA) and glucagon-treated ducklings (103 nmol/kg twice-daily, intraperitoneally, GT). Basal MO2 was higher in CA than in TN or GT ducklings and increased in all groups with elevated flow rates. Catecholamines increased both MO2 and perfusion pressure. The maximal effect on MO2 was higher in CA (+ 36%) and GT ducklings (+ 43%) than in controls (+ 31%), but was associated with reduced vasoconstriction. Flow rate did not consistently potentiate the NE response. At high doses, catecholamines became inhibitory on MO2 while a monotonous increase of pressure was still observed. Glucagon, by contrast, slightly decreased both MO2 and pressure. This vasodilatory effect was greater in CA ducklings than controls in preconstricted preparations. In vivo, low-dose epinephrine induced a modest thermogenic effect (+ 10%) in CA ducklings. These findings showed that duckling muscle thermogenesis is directly stimulated in vitro by catecholamines but not by glucagon. Higher in vitro thermogenic effects of NE in ducklings that were expected to exhibit muscle NST in vivo suggests catecholamine involvement in muscle NST in vivo. Potential vascular control of avian muscle NST is discussed.

Characterization of a cDNA encoding an alpha thyroid hormone receptor in muscovy duckling.

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.

Molecular cloning and sequencing of a cDNA encoding a beta-thyroid hormone receptor in muscovy duckling.

A cDNA clone encoding a beta-thyroid hormone receptor (TRbeta) from muscovy duckling liver was isolated and sequenced. Comparison with the chicken TRbeta sequence showed a high degree of homology. This cDNA was used as a probe to characterize the TRbeta mRNA transcripts expressed in muscovy duckling liver.

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.

Nonshivering thermogenesis in king penguin chicks. I. Role of skeletal muscle.

In cold-acclimatized (CA) king penguin chicks exhibiting nonshivering thermogenesis (NST), protein content and cytochrome oxidase (CO) activity of tissue homogenates were measured together with protein content, CO, and respiration rates of isolated mitochondria from skeletal muscle (gastrocnemius and pectoralis) and liver. The comparison was made with chicks reared at thermoneutrality (TN) for at least 3 wk. In CA chicks showing a NST despite the lack of brown adipose tissue, an increase in thermogenic capacity was observed in skeletal muscle in which the oxidative capacity rose (+28% and +50% in gastrocnemius and pectoralis muscles, respectively), whereas no change occurred in the liver. Oxidative capacity of skeletal muscle increased together with the development of mitochondrial inner membrane plus cristae in muscles of CA chicks contrary to their TN littermates (+30 to +50%). Subsarcolemmal mitochondria of CA chicks had a higher protein content (+65% in gastrocnemius muscle) and higher oxidative capacities than in controls. The lower respiratory control ratio of these mitochondria might result from a low ADP phosphorylation rate. No change occurred in the intermyofibrillar fraction nor in liver mitochondria. These findings together with earlier results obtained in cold-acclimated ducklings indicate the marked and suited adaptation of skeletal muscle and in particular of subsarcolemmal mitochondria allowing them to play a role in NST.

Nonshivering thermogenesis in king penguin chicks. II. Effect of fasting.

The effect of fasting on the energy metabolism of skeletal muscle and liver was investigated in cold-acclimatized short-term fasting (STF) (3 wk) and naturally long-term fasting (LTF) (4-5 mo) king penguin chicks, both groups exhibiting nonshivering thermogenesis (NST). A comparison was made with nourished cold-acclimatized controls. In these chicks, no brown adipose tissue deposits could be found on electron-microscopic observations of fat deposits. Protein content and cytochrome oxidase (CO) activity of tissue homogenates were measured in liver and pectoralis and gastrocnemius muscles, as were protein content, CO activity, and respiration rates of mitochondria isolated from these organs. Fasting-induced protein loss affected the pectoralis more than the gastrocnemius muscle, thus preserving locomotor function. In STF chicks, specific mitochondrial protein content and specific tissue CO activity were preserved but total organ CO capacity was reduced by half in pectoralis and liver following the fall in organ mass. In LTF chicks, both specific and total CO activity were drastically reduced in muscles, whereas specific CO activity was preserved in liver. In these LTF chicks, muscle mitochondria showed an energized configuration associated with an increased area of inner membrane in gastrocnemius. A reduction of respiratory control ratio (RCR) was observed in subsarcolemmal muscle mitochondria of STF chicks, whereas intermyofibrillar and liver mitochondria kept high RCR values.(ABSTRACT TRUNCATED AT 250 WORDS)

Increase in cytochrome oxidase capacity of BAT and other tissues in cold-acclimated gerbils.

To determine the effects of cold acclimation on the oxidative capacity of different tissues and their possible role in nonshivering thermogenesis (NST) in a desert rodent, the gerbil (Gerbillus campestris), measurements of cytochrome oxidase activity (COX) were performed on homogenates of liver, brown adipose tissue (BAT), and subsarcolemmal (SS) and intermyofibrillar (IMF) fractions of skeletal muscle. Total organ mass was also measured. Gerbils were maintained either at thermoneutrality (TN) or cold [4 degrees C, ambient temperature (Ta)] for 4 (CA4) or 8 (CA8) wk. A comparative study was made with mice (Mus musculus). Total and relative masses of BAT increased significantly in both gerbils and mice during cold acclimation, whereas muscle mass decreased in CA4 gerbils. Specific and total COX (TCOX) increased in the three tissues. A considerable increase (+170%, P less than 0.05) of TCOX and protein content in liver of CA4 gerbils compared with controls was observed, whereas no significant changes occurred in liver of CA4 mice. In muscle, an increase of TCOX in SS and a reduction in IMF cell compartments were noted. The increase in BAT oxidative capacity in CA4 gerbils represented only a small portion of that in liver (36%) and in SS fraction of muscle (41%) at the same stage of acclimation. The ability of the three tissues to contribute to components of thermogenesis in vivo was evaluated. The sum of oxidative capacity of the three tissues was largely above the peak metabolic rate (PMR), whereas that of muscle exceeded maximum shivering thermogenesis, which developed both in TN and CA4 gerbils.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonshivering thermogenesis and adaptation to fasting in king penguin chicks.

The ability to develop nonshivering thermogenesis (NST) and the effect of fasting on thermogenic response to cold were studied in winter-acclimatized king penguin chicks. Metabolic rate (MR) and integrated electrical muscle activity were measured at different ambient temperatures. In cold-acclimatized (5 degrees C) fed chicks, shivering threshold temperature (STT) was 9.4 degrees C lower than lower critical temperature (LCT), indicating that NST (0.7 W/kg) occurs at moderate cold, whereas in control chicks fed and reared at 25 degrees C for 3 wk, LCT and STT were similar. Chicks reared in the cold and fasting for 3 wk or 4-5 mo (natural winter fast) developed an NST of 0.8 and 2.4 W/kg, respectively, despite the fast. In fasting chicks, the intercept of the metabolic curve with the abscissa at zero MR was far below body temperature, contrasting with the classic model for heat loss. Their low LCT indicates the capacity of a large reduction in convective conductance characteristic of diving animals and allows energy sparing in moderate cold. Below LCT, conductance reincreases progressively, leading to a steeper than expected slope of the metabolic curve and allowing preservation of a threshold temperature in the shell. These results show for the first time in a wild young bird the development of NST after cold acclimatization. Further, at the temperature of cold acclimatization, an energy-sparing mechanism is shown in response to long-term fast adaptation.

Loose-coupled mitochondria in chronic glucagon-treated hyperthermic ducklings.

In chronic glucagon-treated ducklings (GT) showing thermogenic and hyperthermic responses without shivering to glucagon test injection and in control ducklings (TN; both aged 44 +/- 1 days and reared at thermoneutrality), subsarcolemmal (S) and intermyofibrillar (I) mitochondria from gastrocnemius muscle and mitochondria from liver were isolated. Respiration and cytochrome oxidase activity were determined in these isolated mitochondria by polarography and creatine kinase activity by spectrophotometry, both at 25 degrees C. In GT ducklings, the powerful thermogenesis observed in vivo after a glucagon test injection may be due to the uncoupling effect of released free fatty acids (FFA) in loose-coupled mitochondria because their respiration increased as a function of FFA concentration, and the loose coupling of these mitochondria was reversed by addition of albumin. In all types of mitochondria from GT ducklings, the increase in respiration because of FFA was about double that in mitochondria from controls. There was no change in creatine kinase activity from liver and I mitochondria, but a 16% decrease in this enzyme activity (expressed per mg mitochondrial protein) from S mitochondria was shown despite a strong increase in cytochrome oxidase activity from liver mitochondria (+114% if expressed per g tissue) and from muscle mitochondria (I, +53 or +48%; S, +41 or +97% if expressed per mg mitochondrial protein or per g tissue, respectively). These results support a coupling defect in liver and skeletal muscle mitochondria from the GT hyperthermic ducklings and an uncoupling reinforcement by FFA.

Two daily glucagon injections induce nonshivering thermogenesis in Muscovy ducklings.

In 6-wk-old chronically glucagon-treated (GT) ducklings, the calorigenic effect of intraperitoneal test injection of glucagon was measured at 25 and 4 degrees C ambient temperature (Ta). At 25 degrees C Ta, the increase in metabolic rate (MR) due to test injection of glucagon (360 micrograms/kg) reached 5.3 W/kg (i.e., 98% above the saline control value) in GT ducklings and only 1.7 W/kg (i.e., 29% above the control value) in control (TN) ducklings. After the injection, GT ducklings developed a hyperthermia, reaching 2.4 degrees C, accompanied by intense panting, whereas thermal body temperature did not change in TN ducklings. At 4 degrees C Ta for the same dose of glucagon, no significant change in MR was observed in GT ducklings during 180 min of exposure, whereas a 25% decrease in MR occurred in the same conditions in TN ducklings. In the cold, glucagon injection inhibited shivering in both groups of ducklings but thermogenesis was not suppressed in GT ducklings, showing a true nonshivering thermogenesis in these birds. This nonshivering thermogenesis was estimated to be 3 W/kg (i.e., 55% above resting MR). Such changes produced by chronic glucagon treatment resemble the artificial cold acclimation of rats chronically treated by norepinephrine.

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.

Polygraphic and behavioral study of sleep in geese: existence of nuchal atonia during paradoxical sleep.

In adult geese, chronic polygraphic recordings of EEG, EOG, EMG, ECG and respiratory rate completed with behavioral observations allowed the characterization of four states of vigilance: wakefulness (W), drowsiness (D), slow wave sleep (SWS) and paradoxical sleep (PS). The EEG, EOG, EMG general patterns observed during W, D, SWS and PS episodes with nuchal isotonia or hypotonia were similar to those reported in other birds. The characteristic brevity of avian PS was confirmed since this sleep state occupied only 2.8% of the nycthemere in geese. For the first time in an adult bird it was shown that numerous PS episodes were accompanied, as in mammals, by a total disappearance of nuchal EMG activity. These observations made in a bird species with a stable head support when sleeping, suggest that, as in mammals, inhibitory mechanisms leading to a PS related nuchal atonia do exist and that head falling is not the cause of PS episodes brevity in birds.

Sleep changes in long-term fasting geese in relation to lipid and protein metabolism.

The proportion and the distribution over 24 h of the different arousal stages characterized in geese--wakefulness (W), drowsiness (D), and slow-wave (SWS) and paradoxical sleep (PS)--were studied in caged birds when fed and then fasted during about 40 days. In both the fed and fasted state, each arousal stage was distributed through numerous episodes of a short duration. The geese slept a little more during the night than during the day, a difference that was moderately emphasized during the fast. Fasting induced only a slight decrease in W. There were important changes in SWS and D in relation to the periods of fasting that were previously found to correspond to changes in lipid and protein utilization. When proteins were spared and lipids accounted for most of the energy expenditure, the fasted geese went more and more rapidly from W to SWS (proportion of D decreased from 33 to 13%/day) and slept for progressively longer periods (SWS increased from 23 to 49%/day). By contrast, when protein utilization rose, the general trend in sleep was a decrease in SWS and an increase in D. During the fast, changes in PS were in the same proportion as those in SWS. These reactions are examined for their implications in energy-saving and sleep mechanisms.