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Am J Physiol Regul Integr Comp Physiol 273: R1638-R1648, 1997;
0363-6119/97 $5.00
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Vol. 273, Issue 5, R1638-R1648, November 1997

Hormonal control of thermogenesis in perfused muscle of Muscovy ducklings

Florence Marmonier1, Claude Duchamp1, Frédérique Cohen-Adad1, Tristram P. D. Eldershaw2, and Hervé Barré1

1 Laboratoire de Physiologie des Régulations Energétiques, Cellulaires et Moléculaires, Unité Mixte de Recherches 5578 Centre National de la Recherche Scientifique-Université Claude Bernard Lyon I, Laboratoire Associé Institut National de la Recherche Agronomique, 69622 Villeurbanne, France; and 2 Division of Biochemistry, Faculty of Medicine and Pharmacy, University of Tasmania, Hobart, Australia 7001

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Endocrine stimulation of muscle nonshivering thermogenesis (NST) in ducklings was investigated in vitro using a perfused hindlimb preparation maintained at 25°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°C, TN) were compared with two age-matched groups exhibiting muscle NST in vivo: cold-acclimated ducklings (4°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.

catecholamines; cold acclimation; glucagon; vasoactivity

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

NONSHIVERING THERMOGENESIS (NST) is a common adaptive response to cold found in a number of mammalian species (9) and a few species of birds, including chickens, ducklings, and penguins (2, 13, 19). However, the sites and mechanisms of NST appear to differ in these classes. Brown adipose tissue (BAT) is well known to account for a large proportion of mammalian NST, whereas skeletal muscle is the main site of NST in ducklings (12). BAT thermogenesis is based on an uncoupling of the mitochondrial respiratory chain by the proton-translocator uncoupling protein (28). By contrast, avian mitochondria contain no mammalian-like uncoupling protein (29). Proposed mechanisms for avian NST include those based on fatty acid-induced loose-coupling of mitochondrial respiration (5) and also increased Ca2+ cycling by the sarcoplasmic reticulum (17). Although the sympathetic control of BAT NST is well documented, far less is known about the endocrine control of avian muscle NST.

On the basis of its marked thermogenic and lipolytic effects in birds (3, 4, 6, 14), glucagon appears as a potential mediator of avian NST. Moreover, plasma glucagon concentration is increased in cold-acclimated ducklings (4), and chronic administration of glucagon to ducklings kept at thermoneutrality leads to the development of NST (3). It was postulated that glucagon may trigger muscle NST by stimulating the release of fatty acids from a multilocular adipose tissue differentiated for lipolytic activity (2). Released fatty acids may then affect the respiration of muscle mitochondria, which show a higher basal metabolic rate (MR) and a higher increase in respiration due to the uncoupling effect of fatty acids after cold acclimation (5). As reflected by in vivo measurements of muscle blood flow and arteriovenous differences in oxygen content, muscle NST can be stimulated by exogenous glucagon (14). However, whether the action of glucagon is indirect through lipolysis or direct on myocytes cannot be inferred from these experiments. Besides the action of glucagon, other hormones such as catecholamines could also play a part in the stimulation of avian NST on account of some thermogenic effects of these hormones in birds, both in vivo (6, 24, 31) and in vitro (25). However, such calorigenic action is not invariably found (9) and is much lower than the calorigenic action of glucagon (6).

The use of in vitro perfused muscle preparations has provided numerous insights to the humoral control of muscle metabolism in mammals (reviewed in Ref. 26). Extended studies with perfused hindlimbs in rats have underlined the major role of catecholamines and vasoconstrictors in the modulation of muscle oxygen consumption (11). Recently, with the use of a perfused leg muscle preparation, it has been shown that catecholamines in interaction with glucagon could be potent stimulators of muscle resting oxygen uptake in chickens reared at thermoneutrality (18). It is, however, yet to be established whether this effect is potentiated in animals developing NST and is thus of adaptive interest.

The aim of this experiment was therefore to develop a perfused muscle preparation in ducklings allowing us to address the hypothesis that muscle thermogenesis is under direct hormonal control. Potential thermogenic and vascular effects of catecholamines and glucagon have been investigated in control ducklings reared at thermoneutrality (TN), and also in cold-acclimated (CA) and glucagon-treated (GT) ducklings, the latter two groups of animals showing muscle NST in vivo. Possible calorigenic effects of epinephrine were also investigated in vivo.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals. Male Muscovy ducklings (Cairina moschata L, pedigree R31, Institut National de la Recherche Agronomique) were obtained from a commercial stock breeder (Ets Grimaud). They were fed a commercial mash (Aliment Genthon Démarrage) ad libitum and had free access to water. Ducklings were kept in a constant photoperiod (8:16-h light-dark cycle). The cold-acclimation schedule described by Barré et al. (2) was used: from the age of 1 wk, ducklings were caged in groups of six for a period of 4 wk at either 4°C ambient temperature (Ta) (CA) or 25°C Ta (TN). The glucagon treatment schedule described by Barré et al. (3) was used. From the age of 1 wk, ducklings were caged in groups of six for a period of 4 wk at 25°C Ta and received twice daily an intraperitoneal injection of glucagon (103 nmol/kg; GT).

To standardize the basal values of oxygen consumption and perfusion pressure among the three groups, animals were kept at thermoneutrality (25°C) for 2 h before the start of the surgery.

Birds were cared for under the French Code of Practice for the Care and Use of Animals for Scientific Purposes, and the experimental protocols were approved by the French Ministry of Agriculture Ethics Committee (Animals).

Muscle preparation. The duckling limb surgical preparation was derived from that described by Eldershaw et al. (18) on chicken. Ducklings were anesthetized by continuous halothane inhalation. The left lower limb was surgically isolated and perfused. The major skin vessels were ligated, and the skin covering the limb was carefully removed. A prior intracardiac injection of heparin (2,000 U/kg) prevented blood coagulation during catheterization and the subsequent replacement of blood with artificial perfusate. The popliteal fossa was incised to expose the popliteal artery and vein. The popliteal nerve was divided and the hamstring muscles were ligated and resected proximal to the fossa to give good access for cannulation of the popliteal artery and vein with a polyethylene tubing (1.2 mm ID) filled with heparinized saline. To prevent flow to other tissues, tight ligatures were placed around the ankle and thigh, just above the cannulation site. The duckling was killed with an intracardiac injection of a lethal dose of pentobarbital. The arterial catheter was immediately connected to the perfusion open circuit. The entire surgical procedure routinely lasted ~30 min. During perfusion, the animal was laid on its back and the limb was supported partially aloft. Exposed muscle was covered in plastic cling wrap. After the perfusion had started, the contralateral limb was ligated similarly and excised. The muscles were removed and weighed to allow calculation of required perfusate flow to the perfused limb. This method of estimating perfused muscle mass was validated by infusing 1% Evans blue dye with the perfusate, followed by removal and weighing of stained muscle to determine the mass of muscle perfused, generally in the range of 23-27 g.

Perfusion system. The perfusion system was similar to that described previously (18). Briefly, it consisted of a nonrecirculating constant flow system thermostated at 25°C to ensure adequate oxygenation without the use of red blood cells. A Krebs-Henseleit buffer was used as the perfusion medium, with a composition of (in mM) 120 NaCl; 4.8 KCl; 1.2 KH2PO4; 1.2 MgSO4, 7H2O; 2.5 CaCl2; and 24 NaHCO3; this buffer contained 8.3 mM glucose and 2% bovine serum albumin (BSA), pH 7.4. Before use, the perfusion medium was filtered (Millipore, 0.45 µm). The buffer was continuously gassed with carbogen (95% O2-5% CO2) both in a reservoir placed on ice and by passage through a Silastic tubing lung (5 m; 0.058 cm ID; 0.077 cm OD) before entering the limb muscles to ensure a constant high saturation of arterial O2 and CO2. The whole circuit, which consisted of a heat exchanger, bubble traps, and Silastic lung, was placed in a 25°C thermostated water bath. A small mixing chamber, placed on an electromagnetic stirrer, allowed the injection of hormones directly in the perfusate flow. Buffer solution was pumped from the reservoir through the perfusion system and into the arterial catheter by a Gilson peristaltic pump (Minipuls 8). An in-line pressure transducer (PDCR 75 Nortek Bio 1000) was situated immediately upstream from the arterial catheter. A mercury manometer was used to calibrate the transducer at the start and at the end of a series of experiments. The venous effluent flowed through a 1.5-ml thermostated chamber (25°C) containing a Clark-type oxygen electrode connected to a Gilson apparatus, and the effluent was discarded after measurement of its oxygen partial pressure. The venous tubing and associated electrode chamber did not exert any back pressure on the isolated muscle preparation. The electrode was calibrated before and after each experiment with gas mixture at 100% and 20.93% (atmospheric air) of oxygen. Muscle O2 consumption (MO2) was calculated from arteriovenous difference in O2 content and flow rate; the oxygen dissolution Bunsen coefficient of O2 in plasma was used. Where stated, infusions of hormones into the mixing chamber were made continuously at <1% of the total flow.

Flow rate. Perfusions were conducted at flow rates of either 0.33 ml · min-1 · g-1 (n = 5-6 per group) or 0.47 ml · min-1 · g-1 (n = 5 per group). The lower flow rate was chosen to be within the range of average skeletal muscle blood flow measured in vivo, whereas the higher rate corresponded to an increase in flow equivalent to that seen in vivo after a glucagon injection to the animal (14). The chosen flow rate remained constant throughout each experiment.

Hormone infusion. The effects of infused glucagon and catecholamines [norepinephrine (NE) and epinephrine] were studied. Catecholamines were dissolved in saline (9 g/l NaCl) containing 0.1% ascorbic acid. Solutions were freshly prepared for each experiment. Glucagon solution (Novo-Nordisk) also contained 107 mg lactose/mg glucagon. In each series of experiments, infusion of vehicle alone had no effect on MO2 or perfusion pressure. Hormones were infused in the perfusate flow by a precision peristaltic pump (Gilson minipuls). Final hormonal concentrations tested were between 1 nM and 1 µM.

Muscle metabolites. Muscle metabolite concentrations were determined in limb muscle samples previously freeze clamped with tongs precooled in liquid nitrogen. Samples were obtained after 2 h of perfusion at 25°C at low (0.33 ml · min-1 · g-1) or high (0.47 ml · min-1 · g-1) flow rate with or without hormone stimulation (3-6 determinations per group). Frozen samples were stored at -80°C and powdered under liquid nitrogen. Creatine phosphate, ATP, ADP, and AMP were assayed by spectrophotometric methods. The values obtained with perfused muscles in vitro were compared with in vivo data obtained from a separate batch of ducklings (n = 4-6 ducklings per group). These birds were catheterized (jugular vein) 2 days before sampling under halothane anesthesia. At the time of sampling, ducklings were placed in individual boxes and the catheter was connected to polyethylene tubing. Animals were rested for 2 h and were then anesthetized with 1 ml of pentobarbital sodium (50 mg/kg) injected through the catheter without disturbing the animal. Immediately after anesthesia, limb muscles were exposed and freeze clamped. With this protocol, in vivo samples were obtained with minimal prior contraction of the sampled muscles.

Estimation of edema. Muscle samples were taken from the perfused limb after 2 h of perfusion (n = 3-6 values per group) or from the contralateral limb at the beginning of the experiment (denoted in vivo; n = 4-6 values per group). Samples were dried at 80°C to a constant weight. The ratio of fresh weight to dry weight was calculated from in vivo and perfused muscle samples to estimate the magnitude of edema after 2 h of perfusion with a 2% BSA buffer.

In vivo MR measurement. MR was measured in vivo by indirect calorimetry in an open circuit as described previously (2). To allow saline or epinephrine infusion, the jugular vein was cannulated under halothane anesthesia 2 days before the experiment. On the day of the experiment, the vascular catheter was connected to a continuous infusion pump (A99, Bioblock Scientific), and the bird was positioned in the thermostatic chamber set at thermoneutrality (25°C). As performed with the protocol with perfused muscle in vitro, increasing hormone doses were tested successively in vivo after a 120-min preliminary equilibration period. Each epinephrine infusion lasted 20 min and was followed by a 40-min recovery period. MR was monitored throughout. Total doses (and infusion rates) of epinephrine bitartrate salt were 2 µg/kg (100 ng · kg-1 · min-1), 20 µg/kg (1 µg · kg-1 · min-1), and 100 µg/kg (5 µg · kg-1 · min-1), thus in the range of 6-300 nmol/kg. Epinephrine solutions were freshly prepared immediately before infusion. In vivo experiments were performed with TN (n = 8) and CA (n = 5 or 6) ducklings. Epinephrine was chosen because this hormone induced a higher thermogenic effect than NE in king penguin chicks in vivo (6).

Statistics. Values are presented as means ± SE. Statistical significance of observed variations was assessed by the one-factor analysis of variance (ANOVA) for repeated measures; observed differences between means were then tested by Scheffé's F test. Differences between values from the same group were assessed by Student's paired t-tests. The three-factors ANOVA (hormone concentration × group × flow rate) was reduced to only two factors by calculating the integrated response for the entire range of concentrations tested (area under the curve); a two-way ANOVA was then used. Statistical significance was recognized at P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Characterization of the perfused muscle preparation. Evans blue dye (1%) was routinely infused at the end of perfusion through the arterial catheter to assess the degree of perfusion of the preparation. The resultant staining confirmed that perfusate flow was confined to the lower limb in both hormone-stimulated and nonstimulated preparations. More than 98% of the isolated muscle tissue was actually perfused in this model.

Muscle wet weight-to-dry weight ratio (Table 1) was not different among the three in vivo groups, and no significant change occurred after perfusion at a low flow rate. At the high flow rate, the ratio slightly increased, indicating some edema formation in both TN and CA groups after >2 h of perfusion at this flow rate.

                              
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  		Table 1.   Metabolite concentrations in perfused limb muscle of TN, CA, or GT ducklings

Muscle phosphagen concentrations (Table 1) were no different from in vivo levels after 2 h of perfusion at the low (0.33 ml · min-1 · g-1) or high (0.47 ml · min-1 · g-1) flow rate without hormonal stimulation. Muscle energy charge of the adenylate system, defined as ([ATP] + 0.5 × [ADP])/([ATP] + [ADP] + [AMP]), where brackets indicate concentration, remained at the in vivo value regardless of the perfusion flow rates. Muscle phosphagens concentrations and energy charge were also maintained at the in vivo levels after perfusion with hormonal stimulation. A slight increase in the wet weight-to-dry weight ratio was, however, noted after very high doses of catecholamines.

Basal values of MO2 and perfusion pressure. At the low perfusion rate, basal MO2 was higher in CA (10.1 ± 0.4 µmol · g-1 · h-1, P < 0.01) than in TN (8.1 ± 0.4 µmol · g-1 · h-1) and GT (8.5 ± 0.3 µmol · g-1 · h-1) ducklings (Table 2). Yet the difference between CA and TN ducklings was not significant at the high perfusion flow rate. A significant group effect (P < 0.05) was also observed at low flow rate on basal perfusion pressure, which was higher in TN (43.8 ± 1.6 mmHg) than in CA (35.8 ± 1.1 mmHg) and GT (31.3 ± 1.3 mmHg) ducklings (P < 0.05 in each case). The difference in pressure persisted at the high perfusion flow rate between TN (53.1 ± 2.9 mmHg) and CA (41.3 ± 2.3 mmHg) ducklings (P < 0.05).

                              
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  		Table 2.   Basal values of muscle oxygen uptake and perfusion pressure in TN, CA, or GT ducklings

A flow effect was observed on basal MO2 of TN and CA ducklings (Table 2). An increase in perfusion flow rate induced an increase in MO2 (+32% in TN and +20% in CA ducklings, P < 0.05). The flow effect was therefore slightly more marked in TN than in CA ducklings, leading to a nonsignificant difference in basal MO2 between groups at the high flow rate. The higher flow also induced an increase in perfusion pressure (P < 0.05) in both groups of animals (+21% in TN and +15% in CA animals). At both flow rates and after 30-40 min of stabilization, values of MO2 and pressure remained constant during perfusions without hormone stimulation.

NE effects on MO2 and perfusion pressure. Increasing concentrations of NE, including 1, 10, 20, 50, and 300 nM and 1 µM, were tested. Regardless of prior treatment or flow rate, NE infusion induced a rapid increase in perfusion pressure as well as a rapid decrease in venous PO2 (Fig. 1), indicating a rapid onset of vasoconstriction and an increase in MO2. Effects were sustained throughout the period of NE infusion and were rapidly reversed on cessation of NE infusion. Dose-curve responses for both MO2 and perfusion pressure are shown in Fig. 2. In all treatment groups, NE induced significant increases in MO2 and perfusion pressure at concentrations higher than 1 nM (P < 0.05). Effects on MO2 were higher in CA and GT ducklings than in TN controls, whereas effects on perfusion pressure were lower. The maximal NE-stimulated increase in MO2 at the low flow was 2.5 µmol · g-1 · h-1 in TN (+31% over basal), 3.6 µmol · g-1 · h-1 in CA (+36% over basal), and 3.7 µmol · g-1 · h-1 in GT ducklings (+43% over basal). At higher doses of infused NE (>300 nM), MO2 tended to decrease. The inhibitory effect of high doses of NE was, however, much more marked at the high flow rate, MO2 being significantly decreased in TN ducklings (P < 0.05), whereas it was still increased (+35%) in CA ducklings. In TN animals, the MO2 obtained with the highest dose of NE was not significantly different from the basal value without hormone stimulation. This was despite a dose-dependent continuous increase in perfusion pressure. Perfusion pressures associated with maximal MO2 effects at the low flow rate tended to be higher in TN (+330%) than in CA (+280%) and GT (+260%, P < 0.05) ducklings. Similar trends were observed at the high flow rate. There was a trend for half-maximal effective concentration values to be lower in CA and GT ducklings (3.6 ± 1.7 and 4.1 ± 1.4 nM, respectively) than in TN controls (13.5 ± 1.5 nM).


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  		Fig. 1.   Representative tracings of venous PO2 (top) and perfusion pressure (P, bottom) obtained with perfused leg muscles of a cold-acclimated (CA) duckling. Tracings were recorded at the low flow rate (0.33 ml · min-1 · g-1) with a series of increasing norepinephrine concentrations, which were continuously infused for periods represented by the length of the solid bars.


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  		Fig. 2.   Effects of norepinephrine on muscle oxygen uptake (MO2) and perfusion pressure in thermoneutral (TN), CA, or glucagon-treated (GT) ducklings. Leg muscles were perfused at a low (0.33 ml · min-1 · g-1, left) or high (0.47 ml · min-1 · g-1, right) flow rate. Data are means ± SE from 5 or 6 experiments. In each group, norepinephrine induced a significant increase in both MO2 and P for concentrations >= 10 nM (P < 0.05). When not visible, error bars are within symbols.

Calculations of areas under the curve were used to study the single influence of group and flow rate factors on thermogenic and vascular responses (Fig. 3). A significant group effect was observed on muscle responses to NE, both on MO2 and perfusion pressure. The overall NE-induced increase in MO2 was higher in CA ducklings (+136% at the high flow rate, P < 0.05) and in GT ducklings (+129% at the low flow rate, P < 0.05) than in TN ducklings. Similar results were observed when only the 1- to 10-nM range of NE concentrations was considered (+176% in CA and +157% in GT ducklings, P < 0.05 in each case).


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  		Fig. 3.   Integrated responses to norepinephrine (NE) on MO2 and perfusion pressure in TN, CA, or GT ducklings. Values correspond to the integrated response (area under the curve) obtained with low norepinephrine concentrations (1-10 nM). Values are expressed by difference with basal values in each group (see values in Table 2). Leg muscles were perfused at a low (0.33 ml · min-1 · g-1, open bars) or high (0.47 ml · min-1 · g-1, hatched bars) flow rate. In GT ducklings, experiments were only performed at the low flow rate. Data are means ± SE of 5 or 6 ducklings. Values with different letters are significantly different (P < 0.05).

Although absolute values were higher at the high than the low flow rate, the relative (% of initial value) MO2 increase was similar at both rates, indicating that flow did not potentiate the effect of NE.

Over the entire range of NE doses, the increase in perfusion pressure was lower in CA (high flow, -44%, P < 0.05) and GT (low flow, -43%, P < 0.05) than in TN ducklings. Increases in perfusion pressure were not significantly different in the 1- to 10-nM range of NE concentrations.

Effects of increasing doses of epinephrine (1 nM-1 µM) on MO2 and vasoconstriction in TN (n = 4) and CA (n = 4) ducklings were similar to those observed using NE, with peak values of MO2 obtained at 100 nM and reaching +1.9 µmol · g-1 · h-1 in TN (+20% over basal) and +2.9 µmol · g-1 · h-1 in CA (+28% over basal) ducklings (data not shown). The integrated MO2 response in the 1- to 10-nM range was higher in CA than in TN ducklings (+157%). Higher epinephrine concentrations (>100 nM) were clearly inhibitory, with values at 1 µM being not significantly different from basal levels.

Glucagon effects on MO2 and perfusion pressure. Increasing concentrations of glucagon (0, 1, 10, and 100 nM and 1 µM) were tested on perfused muscles of TN, CA, and GT ducklings (Fig. 4, low flow only). In TN and CA groups, experiments were conducted at the low and high (data not shown) flow rates. After glucagon infusion, a decrease in perfusion pressure was associated with an increase in venous PO2, indicating some vasodilation and a decrease in basal MO2. These effects were usually significant for glucagon concentrations >10 nM (P < 0.05); the decreases in MO2 and perfusion pressure were then proportional to the glucagon concentration tested. The lack of direct thermogenic effect of glucagon was not due to the low perfusion temperature (25°C), because perfusion experiments performed at 40°C showed similar results (data not shown).


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  		Fig. 4.   Effects of glucagon on MO2 and perfusion pressure in TN, CA, or GT ducklings. Leg muscles were perfused at the low (0.33 ml · min-1 · g-1) flow rate. Number of perfusions is given in parentheses. Data are means ± SE. When not visible, error bars are within symbols. In each group, glucagon induced a significant decrease in both MO2 and perfusion pressure for concentrations between 10 and 100 nM (P < 0.05).

The gradual decrease in MO2 after the infusion of increasing concentrations of glucagon was not different in absolute values from one group of animals to the other whatever the perfusion flow rate. By contrast, the decrease in perfusion pressure was more marked when the initial pressure was higher and thus more marked in the TN groups at high (-15%) and low (-12%) flow rate, whereas the decreases were less marked in the CA and GT groups (-2 to -7%)

There was no significant effect of flow rate on the decrease in MO2 caused by glucagon in both TN and CA groups. However, in TN ducklings, the decrease in pressure induced by glucagon was significantly more marked at the high perfusion flow rate (P < 0.05).

Glucagon effects on a preconstricted muscle. The rather small effects of glucagon on basal values of pressure could be due to the fact that muscle preparations were nearly fully vasodilated. Perfused skeletal muscles of TN and CA ducklings were thus preconstricted with 100 nM NE, and the effects of glucagon were tested. Glucagon-induced vasodilation increased with increasing concentrations of the peptide (Fig. 5) and was slightly more marked in CA than in TN ducklings (P < 0.05) with 100 nM glucagon. These effects were nevertheless small compared with the effects of the nitrodilator sodium nitroprusside (0.5 mM), which abolished most of the NE-induced rise in pressure (-75% in TN and -91% in CA), with values becoming not significantly different from the basal without NE. Glucagon had no significant effect on NE-induced MO2, whereas nitroprusside removed most of the effects of NE in TN (-83%) but not in CA ducklings (-68%), in which the MO2 value remained higher (P < 0.05) than the basal.


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  		Fig. 5.   Effects of glucagon (Gluc) and nitroprusside (NP) on norepinephrine-induced increases in MO2 and perfusion pressure in TN (open bars) or CA (solid bars) ducklings. Leg muscles were perfused at the low (0.33 ml · min-1 · g-1) flow rate, and a dose of 100 nM norepinephrine (NE) was used for preconstriction. Data are means ± SE from 4 or 5 experiments. * P < 0.05 vs. effect in TN ducklings.

In vivo effects of epinephrine infusion on MR. Results obtained in vitro prompted us to evaluate the thermogenic effect of catecholamines in vivo. In TN ducklings, epinephrine produced either no significant change in MR at the low dose or a biphasic response at higher doses (Fig. 6). Initially, MR was slightly reduced, followed by an increase in MR that peaked (+29% at 5 µg · min-1 · kg-1, P < 0.05) 10 min after the end of the infusion. In CA ducklings, by contrast, low-dose epinephrine induced a modest but significant increase in MR 10 min after the start of the perfusion, with a peak at 7.5 W/kg (+0.6 W/kg, i.e., +10%, P < 0.05). Increasing the dose of epinephrine did not enhance the response, and at the highest dose, a biphasic response started to appear with no stimulation during the perfusion but a rise in MR afterward.


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  		Fig. 6.   In vivo effects of epinephrine infusion on metabolic rate in TN (bullet ) or CA (open circle ) ducklings at thermoneutrality (25°C). Epinephrine was infused intravenously for 20 min (horizontal bar) at either 100 ng · kg-1 · min-1 (A), 1 µg · kg-1 · min-1 (B), or 5 µg · kg-1 · min-1 (C). Data are means ± SE of 8 TN and 5 or 6 CA ducklings. When not visible, error bars are within symbols. * P < 0.05 vs. resting metabolic rate before epinephrine infusion.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study has investigated for the first time the endocrine control of resting muscle thermogenesis in ducklings. It has been demonstrated that perfusion flow rate and catecholamines can directly stimulate muscle thermogenesis in vitro. Furthermore, the thermogenic effect of catecholamines on perfused muscles in vitro was enhanced in CA and GT ducklings, two groups exhibiting regulatory muscle NST in vivo (2, 3, 12, 14). By contrast, glucagon had no direct thermogenic effect on perfused muscle in vitro. Finally, epinephrine at low doses exerted a small thermogenic effect in vivo in CA ducklings.

Validation of the perfused limb muscle preparation in ducklings. In the present study, the lower limb perfusion preparation originally described in the chicken (18) was successfully applied to ducklings. Perfusions were performed at 25°C to avoid the need for erythrocytes in the perfusate but to allow delivery of sufficient oxygen to the preparation, which was stable with respect to basal MO2, basal perfusion pressure, and high energy phosphate levels throughout the experimental period. Similar studies of muscle metabolism with perfused or incubated mammalian muscle preparations at 25°C gave similar results, with a correction factor, to those performed at 37°C (8, 11). The lower flow rate of 0.33 ml · min-1 · g wet wt-1 of muscle in the popliteal bed was chosen according to microsphere studies of blood flow in birds (12). At this flow rate, the venous PO2 did not fall below 200 mmHg, and muscle phosphagen levels were maintained at their in vivo levels. The energy charge was consequently well preserved, and no effect of increased flow rate on phosphagen levels was observed. Because of the absence of edema development at this low flow rate even after long perfusions, Krebs buffer containing 2% BSA appears to be a suitable perfusate for duckling muscle at low temperatures. At the higher flow rate or after high doses of catecholamines, a slight edema was observed especially in TN ducklings, but similar degrees of edema were reported by others in perfused rat muscles (10). However, this did not cause any rise in basal perfusion pressure over time. The lower vascular resistance of CA and GT ducklings could be partly accounted for by a higher vascular density than in TN animals (15).

Basal values of MO2. The oxygen consumption of resting perfused muscles in TN duckling at 25°C (8.1 ± 0.4 µmol · g-1 · h-1) is similar to that measured in the chicken in the same conditions (7.4 ± 0.3 µmol · g-1 · h-1, Ref. 18). Limb muscles of CA ducklings showed a higher basal MO2 than those of TN ducklings (+25%), although all animals had been submitted to a 2-h fast at thermoneutrality before the surgical preparation. Similarly, the MO2 of incubated skeletal muscle slices from CA sparrows was higher than in controls (1). An increased basal MO2 was also noted in incubated muscles of CA pigs (23), a large mammal devoid of BAT. By contrast, such a difference was not observed with perfused muscles of CA and TN rats (21, 30). This higher basal MO2 of CA vs. TN ducklings could contribute to the higher resting MR measured in vivo at thermoneutrality (Ref. 2 and the present study). It may involve stimulations of several ion-transport mechanisms, such as the membrane Na+-K+-adenosinetriphosphatase (ATPase) activity, as shown in muscles of CA pigs (23), or the sarcoplasmic reticulum Ca2+-ATPase, as demonstrated in muscles of CA ducklings (17). Furthermore, it is in agreement with the increase in mitochondrial basal respiration observed in CA ducklings (5).

Chronic glucagon treatment, by contrast, did not entail any significant modification of basal MO2. Certainly, in whole body studies there is an absence of effect of this treatment on the resting MR measured 24 h after the last injection of glucagon (3). In GT ducklings, exogenous glucagon is required to stimulate muscle NST (3, 14).

Flow-induced increase in MO2. An increase in the perfusion flow rate, within limits compatible with physiological variations, entailed an increase in MO2. It was not caused by an hypoperfusion at the low flow rate because no change in phosphagen levels was induced by the increase in flow, levels remaining similar to those measured in vivo. This phenomenon has already been observed in several perfused muscle preparations in rats and dogs (Ref. 10; review in Ref. 11). This flow-limited O2 uptake can therefore be extended to bird muscles, as shown in ducklings (this study) and in chickens (18). The relationship between MO2 and perfusion flow could be interpreted as a physiological limitation of MO2 at rest by O2 supply (10). Such a phenomenon could possibly account for the heterogeneity of perfusion observed in mammalian muscle at rest, possibly involving vasomotion in terminal arterioles (16). This does not mean that the muscle is hypoxic, because a marked decrease in phosphocreatine concentration should be observed under such circumstances. Heterogeneous perfusion of muscle has, however, not been reported, to our knowledge, in bird muscles. Other mechanisms could also be involved in the flow-induced MO2. Given the link between the increase in MO2 and the rise in perfusion pressure, a direct role of the smooth muscle cells of the vascular network has been suggested (11). Although the contribution of the vascular cell O2 uptake to total MO2 remains to be fully evaluated, it can be noted that the lower flow-induced rise in basal MO2 of CA ducklings was accompanied by a lower rise in perfusion pressure. Alternatively, shear stress-dependent release of autacoids by endothelial cells might have a role in a paracrine control of skeletal muscle metabolism (11). The respective contribution, if any, of these different mechanisms remains unclear.

Thermogenic effect of catecholamines on duckling muscle. A major finding of the present study is the demonstration of a marked thermogenic effect of catecholamines on duckling skeletal muscle in vitro as reflected by the dose-dependent increase in MO2 and vasoconstriction. The dose-response curve over the full concentration range was biphasic, catecholamines stimulating MO2 at low concentrations (<100 nM) but tending to inhibit MO2 at high concentrations (>100 nM). Similar results were obtained after NE infusion in perfused chicken (18) or rat hindlimb (11, 31). The maximal NE-induced increase in MO2 in TN ducklings (+31% over the basal) may be compared with that observed in chickens (+35% over the basal, Ref. 18) and in the rat hindlimb (+60-80%, Refs. 11 and 21). It should be noted that a thermogenic effect of catecholamines in vitro was observed in both constant-flow (11, 30) and constant-pressure (33) perfusions of rat hindlimb. There was no consistent potentiating effect of flow rate on the NE effect in duckling limb muscles, in agreement with the results obtained on rat hindlimb muscles (reviewed in Ref. 11).

The thermogenic effect of NE was observed both at low (1-10 nM) and high doses (>= 100 nM). Low doses are within the physiological circulating levels of hormones observed in birds (22), whereas the high doses may reflect those occurring at sympathetic synapses (reviewed in Ref. 11). Local concentrations near sympathetic nerve terminals may indeed be significantly higher than reported plasma values. Furthermore, some authors have underlined similarities between high NE concentrations (>= 100 nM) and high-frequency sympathetic nerve stimulation (>4 Hz) effects on perfused muscle MO2 and associated perfusion pressure (11). Because skeletal muscle sympathetic nerve activity is stimulated in cold-exposed redpolls (27), high NE concentrations may be reached in skeletal muscles and could play a role in the control of muscle thermogenesis.

Possible involvement of catecholamines in the control of muscle NST. Cold acclimation and chronic glucagon treatment, two treatments inducing duckling muscle NST in vivo (2, 3, 12, 14), were associated with increased thermogenic response to NE. Such a potentiating effect of cold acclimation was not found in rats by Grubb and Folk (21), whereas a marked increase in the thermogenic effect of NE was observed in perfused muscles of CA rats by Shiota and Masumi (30). Contrary to the rat model, in the duckling hindlimb the NE-induced increase in MO2 cannot be due to the presence of diffuse BAT depots in the preparation, because thermogenic BAT is absent in birds (2, 29).

A thermogenic effect of NE at the muscle level in vitro suggests that catecholamines have the potential to mediate some thermogenic effect in vivo. Present results show that epinephrine at low dose exerted a small (+10%) but significant thermogenic effect in CA ducklings. Thermogenic responses to catecholamines in birds in vivo have been sparsely reported with values reaching +10-40% over basal MR (6, 24, 31). Furthermore, a higher thermogenic response to NE was reported in CA than in warm-acclimated pigeons (24). This is in keeping with observations that plasma catecholamine concentrations is higher in CA than in TN birds (see Ref. 6 for references) and that the sympathetic nervous system is activated in cold-exposed birds, as demonstrated by an increased tissue NE turnover (27). However, it contrasts with many studies that did not find any thermogenic effect of catecholamines in birds (reviewed in Ref. 9). Such discrepancy could result from differences between species or from the use of high concentrations of hormone resulting in responses corresponding to the inhibitory part of the in vitro dose-response curve associated with excessive vasoconstriction. Present results indeed indicate that the response to epinephrine in vivo depends on the cold-acclimation status of the bird and the dose used. At the high dose, the biphasic response, which is similar to that observed with catecholamines in penguins (6), suggests that the thermogenic effect of catecholamines in birds in vivo may be limited by their vasomotor action. The potential for catecholamine-induced muscle NST shown in vitro may therefore be expressed in vivo when their vasomotor action is reduced, such as in CA ducklings.

In the present experiments, high concentrations of catecholamines decreased MO2 in constant-flow perfused duckling limb, especially in TN animals. Such inhibition was not observed in CA and GT ducklings, possibly because of a lower increase in perfusion pressure. An extended resistance capacity to vasoconstrictive and thermogenic effects of high doses of NE has also been reported in the hindlimb of CA rats (21). It is therefore possible that the inhibiting effect on MO2 of high doses of NE in TN ducklings is related to the marked vasoconstriction observed, somehow creating functional arteriovenous shunts in the absence of anatomically identified shunts (11). Similar pressure-related shunts have been suggested in rat hindlimb infused with serotonin or with high doses NE (11).

The higher thermogenic effect of NE in vitro in ducklings that display muscle NST in vivo suggests a possible key role of catecholamines in the activation of muscle NST in vivo. It should, however, be noted that the catecholamine-induced thermogenic effect in vivo in CA ducklings is rather modest (+10%) as compared with the potential for NST estimated in vivo (+71%) by simultaneous measurements of MR and electromyographic activity in cold-exposed CA ducklings (2). Clearly, NST induced by exogenous infusion of catecholamines does not reach the magnitude of cold-induced muscle NST. A similar conclusion was pointed out previously with glucagon-induced calorigenic effect, which cannot account for the entire cold-induced NST (14). It is therefore possible that several factors are acting together in vivo to activate muscle NST.

Glucagon effects on MO2 and perfusion pressure. On the basis that the higher muscle thermogenic response to catecholamines observed after cold acclimation was reproduced by the chronic administration of glucagon at thermoneutrality (GT ducklings), the present results support assertions that glucagon may play a major role in the long-term development of muscle NST in birds (3, 6). This is in keeping with other results obtained in vivo suggesting that glucagon could be a potential mediator of avian muscle NST (3, 6, 14). It was, however, not known whether the short-term thermogenic effect of glucagon was direct on skeletal muscle cells or mediated indirectly for instance by the release of endogenous stimulators such as fatty acids.

The present results obtained in vitro demonstrate that glucagon does not directly stimulate MO2. In fact, with the constant-flow perfusion system used, glucagon alone acted to decrease basal MO2 in all treatment groups. Because glucagon also showed vasodilatory effect, this hormone is thus acting in a fashion similar to most other vasodilators, i.e., by decreasing the MO2 of constant flow-perfused muscles, possibly by dilating nonnutritive vessels to increase perfusion heterogeneity (11). This effect, which was low on basal perfusion pressure, presumably because the limb was virtually fully dilated, was enhanced in an NE-preconstricted preparation, especially in CA ducklings. In these experimental conditions, the vasodilatory effect of glucagon differed from that obtained with nitroprusside because it was not accompanied by a drop in MO2, suggesting that glucagon also affects nutritive flow.

The present results therefore indicate that the action of glucagon as a potential mediator of muscle NST is likely to be indirect, possibly via its vasomotor, metabolic, and neurogenic actions. The vasodilatory effect of glucagon may stimulate MO2 in vivo. Indeed, because glucagon both increases cardiac output in the whole animal (14) and induces a concomitant relaxation of blood vessels (present study), it would cause increased flow to skeletal muscles. Such a rise in muscle blood flow after glucagon has already been measured using the microsphere technique (14), and the present results show that an increased flow to skeletal muscle increases MO2 (Table 2). It is, however, likely that this effect does not account for the entire glucagon-induced thermogenesis observed in vivo. Alternatively, the vasodilatory effects of glucagon may act to potentiate the thermogenic effects of endogenous catecholamines by lowering the detrimental effects of vasoconstriction (18). Besides its cardiovascular effects, glucagon has also marked effects on the mobilization of lipids and carbohydrates, which could possibly modulate muscle thermogenesis. Finally, recent data from this laboratory have shown that glucagon injection stimulates the endogenous release of catecholamines in ducklings (Y. Filali-Zagzouti, H. Abdelmelek, J.-R. Pequignot, and H. Barré, unpublished data), suggesting that part of the glucagon effect in vivo may be mediated by catecholamines. This is in keeping with an activation of muscle sympathetic nerve activity, which has already been shown in man (32). Clarification of these different possibilities obviously deserves investigation.

Possible involvement of the vascular system in muscle metabolic responses. The observation of a thermogenic effect of catecholamines in duckling muscles in vitro raises the question of the possible mechanism involved. Although increased ion cycling at the myocyte level has been suggested on account of the inhibitory effect of ouabain on the NE-induced effect in CA rats (30), no clear mechanism has emerged in mammals. It has, however, become clear that the NE-induced muscle thermogenesis may involve a vascular control of perfused muscle metabolism (11). This is based on the close relationship between O2 and perfusion pressure observed in perfused rat muscle. In ducklings, similar linear relationships were observed between changes in MO2 and increases in pressure at low doses of NE (r = 0.6-0.8 in the three groups, P < 0.05). However, increases in MO2 were greater in CA and GT ducklings, the groups exhibiting the lowest increases in pressure. This result does not directly support a major role for contracting vascular smooth muscle cells as the primary site of O2 uptake (11), although any such relationship is likely to be complex in perfused muscle preparations. Vasoconstriction, however, does appear to have an essential role in NE-induced MO2 because nitroprusside, a dilator specific to vascular smooth muscle, abolished the NE-induced rise in pressure and removed most of the effects on MO2. Similar results were also noted in rat and chicken perfused muscles (11, 18). It has been proposed that the vascular system may act to control the supply of nutrients to putative regions of increased thermogenesis within the muscle (11). Although specialized heat-producing muscle cells have been described in a modified eye muscle of some fishes (7), no such specialization has been reported in avian skeletal muscle. However, decreases in the coupling state of mitochondria of the slow-twitch fiber types have been reported in ducklings (15), suggesting increased thermogenic capacity of these fibers. Interestingly, there is a higher density of resistance arterioles in slow compared with fast mammalian muscles (reviewed in Ref. 11), suggesting that flow to slow fibers might be more sensitive to vascular effects. It should now be investigated whether catecholamines induce a redistribution of perfusate flow in duckling skeletal muscle and whether any such effect is altered by either cold acclimation or glucagon treatment.

In conclusion, we have directly assessed the in vitro endocrine modulation of muscle NST in ducklings by using a constant flow-perfused preparation. Contrary to glucagon, catecholamines can stimulate muscle thermogenesis in vitro, and the effect was enhanced after cold acclimation. Long-term administration of glucagon to ducklings reared at thermoneutrality reproduced the effects of cold acclimation, further supporting a role for this peptide in the cold acclimation process in birds. The higher thermogenic effect of NE in vitro in ducklings that display muscle NST in vivo raises the possibility of catecholamine involvement in activating this mechanism. A potential role of the vasculature in the control of avian muscle NST is also suggested.

Perspectives

Present results shed new light on the endocrine modulation of muscle NST in birds and emphasize the potent role played by the vasculature in modulating MO2. The implications of these in vitro data with respect to the in vivo situation are unclear but raise the possibility that excessive vasoconstriction may be detrimental to the thermogenic capacity of skeletal muscle. This may be the reason why little if any catecholamine-mediated thermogenic effect is observed in vivo in birds. The potential for catecholamine-induced muscle NST as demonstrated in vitro may be expressed in vivo when vasomotor action is reduced, such as in CA ducklings. In the rat, exercise capacity and muscle insulin sensitivity are also altered by vasomotion (reviewed in Ref. 11), suggesting that vasomotor modulation of muscle metabolism may be a general phenomenon. The biochemical mechanism underlying catecholamine-induced thermogenesis in skeletal muscle is currently unclear. The thermogenic effects of catecholamines in duckling perfused muscles (i.e., in animals devoid of BAT) do not support the possibility that the similar results obtained in rats are related to the presence of BAT in the preparation. Thus skeletal muscle itself may be a site of catecholamine-induced thermogenesis. The recent discovery of a gene closely related to uncoupling protein and expressed in rat skeletal muscle (20) may be directly related to the observed regulatory thermogenic capacity of this tissue. Consequently, the potential presence of such a protein in birds, especially those exhibiting skeletal muscle NST, should be investigated in an attempt to define a mechanism for the thermogenic effect of catecholamines in skeletal muscle.

    ACKNOWLEDGEMENTS

Authors are greatly indebted to M. G. Clark for helpful discussions and to E. Q. Colquhoun for invaluable help, suggestions, and discussions in setting up the perfused muscle preparation.

    FOOTNOTES

This work was supported by grants from the Université Claude Bernard, the Institut National de la Recherche Agronomique (INRA), and the Centre National de la Recherche Scientifique (CNRS). F. Marmonier was in receipt of a Région Rhône-Alpes fellowship.

Address for reprint requests: C. Duchamp, Lab. Physiologie des Régulations Energétiques, Cellulaires et Moléculaires, UMR 5578 CNRS, LA INRA, Fac. Sciences, Bât 404, 4ème étage, 43 Bld 11 Novembre 1918, F-69622 Villeurbanne Cedex, France.

Received 13 August 1996; accepted in final form 3 July 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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