J Comp Physiol B DOI 10.1007/s00360-016-0975-3
ORIGINAL PAPER
Lipid‑induced thermogenesis is up‑regulated by the first cold‑water immersions in juvenile penguins Loïc Teulier1 · Benjamin Rey2 · Jérémy Tornos1 · Marion Le Coadic1 · Pierre‑Axel Monternier1 · Aurore Bourguignon1 · Virginie Dolmazon1 · Caroline Romestaing1 · Jean‑Louis Rouanet1 · Claude Duchamp1 · Damien Roussel1
Received: 11 December 2015 / Revised: 1 February 2016 / Accepted: 16 February 2016 © Springer-Verlag Berlin Heidelberg 2016
Abstract The passage from shore to marine life is a critical step in the development of juvenile penguins and is characterized by a fuel selection towards lipid oxidation concomitant to an enhancement of lipid-induced thermogenesis. However, mechanisms of such thermogenic improvement at fledging remain undefined. We used two different groups of pre-fledging king penguins (Aptenodytes patagonicus) to investigate the specific contribution of cold exposure during water immersion to lipid metabolism. Terrestrial penguins that had never been immersed in cold water were compared with experimentally cold-water immersed juveniles. Experimentally immersed penguins underwent ten successive immersions at approximately 9–10 °C for 5 h over 3 weeks. We evaluated adaptive thermogenesis by measuring body temperature, metabolic rate and shivering activity in fully immersed penguins exposed to water temperatures ranging from 12 to 29 °C. Both never-immersed and experimentally immersed penguins were able to maintain their homeothermy in cold water, exhibiting similar thermogenic activity. In vivo, perfusion of lipid emulsion at thermoneutrality induced a twofold larger calorigenic response in experimentally immersed than in never-immersed birds. In vitro, the respiratory rates Communicated by G. Heldmaier. * Damien Roussel damien.roussel@univ‑lyon1.fr 1
Laboratoire d’Ecologie des Hydrosystèmes Naturels et Anthropisés, UMR 5023 CNRS, Université Claude Bernard Lyon 1, ENTPE, Lyon, Bâtiment Charles Darwin C, 69622 Villeurbanne cedex, France
2
Laboratoire de Biométrie et Biologie Evolutive, UMR 5558 CNRS, Université Claude Bernard Lyon 1, 69622 Villeurbanne cedex, France
and the oxidative phosphorylation efficiency of isolated muscle mitochondria were not improved with cold-water immersions. The present study shows that acclimation to cold water only partially reproduced the fuel selection towards lipid oxidation that characterizes penguin acclimatization to marine life. Keywords Metabolism · Mitochondria · Skeletal muscle · Energy substrates · Oxidative phosphorylation efficiency
Introduction Survival of endotherms in cold water is limited by the tremendous energetic lost imposed by the 23-fold higher conductance of water than air of the same temperature. This energetic constraint would be increased by locomotion, which may further enhance the cooling power of water by increasing convective heat losses. Some endothermic species, such as king penguins (Aptenodytes patagonicus) spend most of their living time foraging in the cold circumantarctic ocean (Charrassin and Bost 2001; Pütz and Cherel 2005). Penguin capacity to withstand prolonged expenditure of energy relies on their ability to reduce heat losses and sustain high levels of heat production for long periods (Barré and Roussel 1986; Teulier et al. 2012). However, the energetic constraints triggering the development of such thermogenic capacity when penguin juveniles depart to sea remain unknown. King penguins return on shore every spring to molt and breed. From hatching to fledging time, which occurs approximately after 12–14 months, king penguin chicks are strictly terrestrial, until they molt to acquire waterproof feathers (Cherel et al. 2004). At fledging time, penguin juveniles must face the tremendous energetic demand of
13
marine life to sustain thermoregulatory needs in cold sea water, long distance swimming to reach their fishing area (Péron et al. 2012; Charrassin and Bost 2001) and deep diving (from 50 to 300 m) to catch their prey (Kooyman et al. 1982; Pütz and Cherel 2005). Although local hypothermia and a powerful peripheral vasoconstriction during diving bouts are mechanisms by which penguins minimize heat losses and metabolic cost of thermoregulation (Dumonteil et al. 1994; Handrich et al. 1997), the overall daily field metabolic rate of penguins remains two to four times the shore resting metabolic rate at sea (Kooyman et al. 1982; Nagy et al. 2001; Froget et al. 2004). In line, experimental immersion into moderately cold water (8–10 °C) increases up to threefold the metabolic rate of penguins (Barré and Roussel 1986; Stahel and Nicol 1988; Dumonteil et al. 1994; Bevan et al. 1995; Fahlman et al. 2005). Therefore, the development of adaptive responses during the transition from shore to marine life represents a key step in the nutritional emancipation of penguin juveniles. Skeletal muscles are involved in locomotion and appear as the main source of regulatory thermogenesis in coldacclimated birds, producing metabolic heat by shivering and non-shivering mechanisms (Hissa 1988; Duchamp et al. 1999; Swanson and Vézina 2016). But, to be fully effective, muscle metabolism must be fuelled by commensurate energy substrate delivery. In birds, lipids are by far the main mobilized substrate for fuelling avian muscles at a high rate as it is the case during flight migration or cold acclimation (Dawson et al. 1983; Bénistant et al. 1998; Bedu et al. 2002; Vaillancourt et al. 2005; Weber 2009; Guglielmo 2010). Fatty acids also play a major role in regulatory thermogenesis by acting as stimulators of muscle thermogenic mechanisms in birds (Duchamp et al. 1999; Toyomizu et al. 2002; Talbot et al. 2004; Rey et al. 2010). Recently, we reported that lipids infusion in vivo induced a fourfold larger thermogenic effect in sea-acclimatized immature king penguins than in pre-fledging juveniles (Teulier et al. 2012). A high level of heat production that was associated with (1) a selective upregulation of lipid handling and oxidation in skeletal muscle (Teulier et al. 2012), and (2) a greater fatty acid-induced uncoupling of oxidative phosphorylation in muscle mitochondria (Talbot et al. 2004). However, the underlying energetic constraint responsible for the coordinated increases in lipid oxidation and thermogenic activity reported in sea-acclimatized penguins remains uncertain, as the effects of cold and exercise are difficult to discriminate. Indeed, cold acclimation leads to adaptive changes in skeletal muscles that resemble those resulting from endurance exercise, such as an increased muscle masses, capillary density and oxidative capacity and a shift toward slow-oxidative muscle fiber types (Duchamp et al. 1991, 1992; Mathieu-Costello et al. 1998; Ueda et al. 2005; Zhang et al. 2015). Furthermore, both
13
J Comp Physiol B
cold and exercise training trigger a coordinated increase in fatty acid supply to skeletal muscles (Dawson et al. 1983; Bénistant et al. 1998; Guglielmo 2010). The aim of this study was to disentangle the specific role of cold-water immersion from other aspects of sea acclimatization (swimming, diving) in the improvement of lipid catabolism and thermal ability. For this purpose, we simulated the passage to marine life by ten successive immersions in cold water in pre-fledging king penguin juveniles (Barré and Roussel 1986). We evaluated the capacity for adaptive thermogenesis by simultaneously measuring whole-body metabolic rate and shivering in penguins acutely exposed to different water temperatures. Finally, we evaluated the metabolic adjustments of lipid metabolism (1) in vivo by measuring the responses of metabolic rate and plasma metabolite concentrations to lipid infusion; and (2) in vitro by investigating muscle mitochondrial bioenergetics. Experimentally immersed penguins were compared to never-immersed pre-fledging juveniles.
Materials and methods Animals Field experiments were conducted on the Crozet archipelago (Possession Island, 46°25′ S, 51°45′ E) at the French Alfred Faure Station during six austral summer campaigns. According to the Agreed Measures for the Preservation of Antarctic and Sub-Antarctic Fauna, the project received the ethical approval of the Committee for the French Polar Research Institute (IPEV; program no. 131). King penguin juveniles (Aptenodytes patagonicus) of both sexes (12–14 months old) were captured on the nearby breeding colony of Baie du Marin before they had completed molting which is a pre-requisite for departing to sea. Captured birds finished their molt in an outside enclosure near the laboratory. Birds were weighed daily and force fed on Atlantic mackerel (Scomber vernalis). The amount of food (from 350 to 700 g per bird) was adjusted daily for each individual to maintain body mass. Half of the birds was fed for 3–5 days and then fasted for 48 h before experiments; they constituted the never-immersed (NI) group. In a second group, penguins were exposed to ten cold-water immersions at approximately 9–10 °C for 5 h per immersion every second day as previously described (Barré and Roussel 1986; Talbot et al. 2004). Noted that this water temperature was at approximately 5 °C warmer than temperatures usually undergone by king penguins in the austral Ocean (Charrassin and Bost 2001; Péron et al. 2012). Penguins were fed on a daily basis during the whole immersion protocol and then fasted for 48 h before experiments; they constituted the immersed (IM) group. On
J Comp Physiol B
completion of the study, all penguins were released at the site of their capture. Thermogenic activity in cold water Two successive austral summer campaigns (December– January 2005–2006 and 2006–2007) were devoted to simultaneously measure energy expenditure (indirect calorimetry) and shivering activity (electromyographic activity, EMG) of fully immersed penguin in vivo, at different water temperatures ranging from 29 to 12 °C. Twenty two birds were included in this protocol (12 NI and 10 IM weighing on average 8.2 ± 0.2 and 8.1 ± 0.2 kg, respectively). Penguins were placed in a thermostatic chamber consisting of an insulated 70-l polythene tank and left to equilibrate for 1 h at 10 °C in air before metabolic rate was monitored for 15 min. Then the tank was filled with water at 29 °C, the highest water temperature tested in the present protocol. To obtain metabolic steady state and thermal equilibrium, there was a 1 h adjustment period before the recording metabolic procedure was repeated. Thereafter, water temperature was reduced by steps, and after 1.5 h necessary to reach thermal equilibrium and metabolic steady state, the metabolic rate was measured over 15 min by indirect calorimetry using an open-circuit system (Barré and Roussel 1986; Teulier et al. 2012) at water temperatures ranging from 29 to 12 °C. Water temperature within the tank was monitored using a digital set-point potentiometric temperature controller coupled with a heater–cooler exchanger and a water-circulating pump. Body (stomach) temperature was continuously monitored during the experiment with a copper-constantan thermocouple. Shivering was measured as EMG activity of the pectoralis muscle. The EMG signal received from three monopolar electrodes insulated except for the tips (Stabilohm 110, Ni 80 % and Cr 20 %, 0.12 mm diameter) and acutely inserted into the muscle 10 mm apart was monitored on MP30B-CE, Biopac System and recorded with Biopac Student Lab Pro v.3.6.7 software (Santa Barbara, CA, USA) as previously described (Teulier et al. 2010). Catheterization and in vivo lipid oxidation One austral summer campaign (November–January 2008– 2009) was devoted to measure the metabolic effect of a triglyceride infusion in vivo. This protocol was performed at 10 °C (in air) within the thermoneutral zone for these birds (Froget et al. 2002). Eleven birds were included in this protocol (5 NI and 6 IM weighing on average 9.2 ± 0.2 and 8.0 ± 0.3 kg, respectively). Birds were catheterized and after stabilization within the thermostatic chamber for a night, metabolic rate was continuously recorded by indirect calorimetry following 30-min infusions at 1 mL/min
of saline (0.9 % NaCl) then Intralipid 20 % emulsion (Fresenius Kabi AB, Sweden) as described previously (Teulier et al. 2012). The day before experimentation, birds were equipped with intravenous catheters in marginal vein of each flipper as described previously (Teulier et al. 2012). Birds were thereafter placed in a thermostatic chamber at thermoneutrality during the night to ensure their complete recovery. The day of experiment, the first catheter (Braun Introcan 20G¼, 1.1 × 32 mm) was connected to a calibrated syringe pump (Kd Scientific, model 100, serial 931, USA) infusing continuously at 1 mL/min a saline solution (0.9 % NaCl) for 30 min. Eighty min after the end of saline infusion, a triglyceride emulsion (Intralipid® 20 %, Fresenius Kabi AB, Sweden) was infused during 30 min. The metabolic rate was continuously monitored (starting 45 min before the saline infusion and ending 2 h and a half after the end of triglyceride infusion) at 10 °C in air within the thermoneutral zone of these birds (Barré and Rouanet 1983). The in vivo total effect of triglyceride infusion upon metabolic rate was estimated by calculating the area under the curve (AUC) for each bird using trapezoidal integration minus the individual resting metabolic rate (Teulier et al. 2012). Blood sampling and kinetic parameters of metabolite disappearance The second catheter (BD Insyte-W 16G, 1.7 × 45 mm) was extended with 12 cm of Silastic® (Dow Corning, 0.64 × 1.19 mm, USA) to lessen the impact of vasoconstriction during blood sampling. Blood samples (500 µL) taken at different time points were centrifuged at 3500×g for 5 min at 4 °C and the resulting plasma samples stored at −80 °C until analysis of plasma triglyceride (TG), nonesterified fatty acid (NEFA), and glycerol concentrations using commercially available kits from Biomérieux (TG and glycerol) or Wako chemicals (NEFA). Kinetic parameters of plasma lipids were calculated using the first-order disappearance rate constant (k) obtained from linear regression of the logarithm of metabolite concentration against time data from the maximum metabolite concentration (Cmax) measured at the end of infusion period. The rate of disappearance (Rd) was calculated as (Rd = Cmax × k). In vitro mitochondrial respiration Two austral summer campaigns (December–January 2004–2005 and 2010–2011) were devoted to biochemical analysis of muscle mitochondria in vitro. Twenty birds were included in this protocol (14 NI and 6 IM weighing on average 7.8 ± 0.1 and 7.7 ± 0.2 kg, respectively). Superficial pectoralis muscle was surgically biopsied under general isoflurane anesthesia as described previously (Talbot
13
et al. 2004; Rey et al. 2008). The biopsy (1 g) was freshly used for mitochondria extraction and bioenergetics analysis. Intermyofibrillar and subsarcolemmal mitochondrial populations were isolated by a standard extraction protocol, involving potter homogenization, partial protease digestion and differential centrifugations (Talbot et al. 2004; Rey et al. 2008; Teulier et al. 2012). Mitochondria were pelleted at 8700×g and the amount of mitochondrial proteins was determined by a Biuret method with bovine serum albumin as standard. Mitochondrial oxygen consumption was measured using a Clark oxygen electrode (Rank Brothers LTD, Cambridge, UK) maintained at 38 °C and calibrated with an air-saturated respiratory medium [120 mM KCl, 5 mM KH2PO4, 1 mM MgCl2, 1 mM EGTA, 3 mM Hepes (pH 7.4) and 0.3 % (w/v) fatty acid-free bovine serum albumin]. Respiration was measured using NADH-linked substrates derived either from carbohydrate metabolism (5 mM pyruvate/2.5 mM malate) or lipid metabolism (40 µM palmitoyll-carnitine/2.5 mM malate), or a FADH2-linked substrate derived from citric acid cycle (5 mM succinate in the presence of 5 µM rotenone). The basal non-phosphorylating respiration rate (state 4oligo) was measured in the presence of 2.5 µg/mL oligomycin. The maximal fully uncoupled respiration rate (state FCCP) was initiated by the addition of 2 µM carbonyl cyanide p-tri-fluoro-methoxy-phenyl-hydrazone (FCCP). The respiratory control ratio (RCR) refers to the ratio of FCCP-induced maximal respiration rate (state FCCP) to basal non-phosphorylating respiration rate measured in the presence of oligomycin (state 4oligo). Mitochondrial oxidative phosphorylation efficiency One austral summer campaign (December–January 2013– 2014) was devoted to the biochemical analysis of muscle mitochondria in vitro. Fifteen birds were included in this protocol (8 NI and 7 IM weighing on average 9.3 ± 0.2 and 9.0 ± 0.3 kg, respectively). Superficial pectoralis muscle was surgically biopsied under general isoflurane anesthesia as described previously (Talbot et al. 2004; Rey et al. 2008). The biopsy (0.5 g) was freshly used for mitochondria extraction and bioenergetics analysis. Mitochondrial populations were isolated by a standard extraction protocol, involving potter homogenization, partial protease digestion and differential centrifugations (Monternier et al. 2014). The mitochondrial oxidative phosphorylation efficiency was assessed at 38 °C by measuring the rates of oxygen consumption and ATP synthesis in the respiratory medium supplemented with glucose (20 mM), hexokinase (1.5 U/ mL) and different concentrations of ADP ranging from 5 to 100 µM as described previously (Teulier et al. 2010; Monternier et al. 2014). Respiration was initiated by adding a mixture of respiratory substrates consisting of pyruvate (5 mM), malate (2.5 mM) and succinate (5 mM). Kinetics
13
J Comp Physiol B
parameters of mitochondrial oxidation, i.e., the apparent affinity (Km) for ADP and the maximal oxidation rate (Vmax) of mitochondria, were determined from the dependence of mitochondrial oxygen consumption rate on ADP concentration ranging from 5 to 500 µM. Statistical analysis Relations between integrated EMG activity and water temperatures, or metabolic rate and water temperatures were expressed by two linear regression lines (Teulier et al. 2010) that intersect at the shivering threshold temperature (STT) or at the lower critical temperature (LCT), respectively. To draw these regression lines, we statistically determined by a paired-ANOVA test at which ambient temperature EMG activity or metabolic rate became significantly different from basal values, respectively. These values and those measured at lower water temperatures were then integrated in a second linear regression line distinct from the basal linear regression line. Two-way repeated-measure analyses of variances (RM ANOVA) followed by protected least significant difference tests were performed to estimate the effects of groups and infusions on metabolic rate and metabolite kinetics (SigmaPlot v.12, Systat Software, Inc., San Jose, CA, USA). When the assumptions of normality (Shapiro–Wilk test) or homoscedasticity (Levene test) were not met, Friedman RM ANOVA on ranks was used. Mitochondrial apparent Km for ADP and Vmax was determined for oxygen consumption by fitting experimental data by the Michaelis–Menten equation: V = [Vmax × (ADP)]/ [Km + (ADP)] using sigma plot 12.0 software. Two-way repeated-measure analyses of variances (RM ANOVA) followed by protected least significant difference tests were performed to estimate the effects of groups and ADP addition on mitochondrial respiratory rates (SigmaPlot v.12, Systat Software, Inc., San Jose, CA, USA). Mitochondrial oxidative phosphorylation and kinetic parameters were tested with analysis of variance (ANOVA) for independent values, followed by protected least significant difference tests (Statview v4.5 software). All data are presented as mean ± SEM with significance considered at p
ORIGINAL PAPER
Lipid‑induced thermogenesis is up‑regulated by the first cold‑water immersions in juvenile penguins Loïc Teulier1 · Benjamin Rey2 · Jérémy Tornos1 · Marion Le Coadic1 · Pierre‑Axel Monternier1 · Aurore Bourguignon1 · Virginie Dolmazon1 · Caroline Romestaing1 · Jean‑Louis Rouanet1 · Claude Duchamp1 · Damien Roussel1
Received: 11 December 2015 / Revised: 1 February 2016 / Accepted: 16 February 2016 © Springer-Verlag Berlin Heidelberg 2016
Abstract The passage from shore to marine life is a critical step in the development of juvenile penguins and is characterized by a fuel selection towards lipid oxidation concomitant to an enhancement of lipid-induced thermogenesis. However, mechanisms of such thermogenic improvement at fledging remain undefined. We used two different groups of pre-fledging king penguins (Aptenodytes patagonicus) to investigate the specific contribution of cold exposure during water immersion to lipid metabolism. Terrestrial penguins that had never been immersed in cold water were compared with experimentally cold-water immersed juveniles. Experimentally immersed penguins underwent ten successive immersions at approximately 9–10 °C for 5 h over 3 weeks. We evaluated adaptive thermogenesis by measuring body temperature, metabolic rate and shivering activity in fully immersed penguins exposed to water temperatures ranging from 12 to 29 °C. Both never-immersed and experimentally immersed penguins were able to maintain their homeothermy in cold water, exhibiting similar thermogenic activity. In vivo, perfusion of lipid emulsion at thermoneutrality induced a twofold larger calorigenic response in experimentally immersed than in never-immersed birds. In vitro, the respiratory rates Communicated by G. Heldmaier. * Damien Roussel damien.roussel@univ‑lyon1.fr 1
Laboratoire d’Ecologie des Hydrosystèmes Naturels et Anthropisés, UMR 5023 CNRS, Université Claude Bernard Lyon 1, ENTPE, Lyon, Bâtiment Charles Darwin C, 69622 Villeurbanne cedex, France
2
Laboratoire de Biométrie et Biologie Evolutive, UMR 5558 CNRS, Université Claude Bernard Lyon 1, 69622 Villeurbanne cedex, France
and the oxidative phosphorylation efficiency of isolated muscle mitochondria were not improved with cold-water immersions. The present study shows that acclimation to cold water only partially reproduced the fuel selection towards lipid oxidation that characterizes penguin acclimatization to marine life. Keywords Metabolism · Mitochondria · Skeletal muscle · Energy substrates · Oxidative phosphorylation efficiency
Introduction Survival of endotherms in cold water is limited by the tremendous energetic lost imposed by the 23-fold higher conductance of water than air of the same temperature. This energetic constraint would be increased by locomotion, which may further enhance the cooling power of water by increasing convective heat losses. Some endothermic species, such as king penguins (Aptenodytes patagonicus) spend most of their living time foraging in the cold circumantarctic ocean (Charrassin and Bost 2001; Pütz and Cherel 2005). Penguin capacity to withstand prolonged expenditure of energy relies on their ability to reduce heat losses and sustain high levels of heat production for long periods (Barré and Roussel 1986; Teulier et al. 2012). However, the energetic constraints triggering the development of such thermogenic capacity when penguin juveniles depart to sea remain unknown. King penguins return on shore every spring to molt and breed. From hatching to fledging time, which occurs approximately after 12–14 months, king penguin chicks are strictly terrestrial, until they molt to acquire waterproof feathers (Cherel et al. 2004). At fledging time, penguin juveniles must face the tremendous energetic demand of
13
marine life to sustain thermoregulatory needs in cold sea water, long distance swimming to reach their fishing area (Péron et al. 2012; Charrassin and Bost 2001) and deep diving (from 50 to 300 m) to catch their prey (Kooyman et al. 1982; Pütz and Cherel 2005). Although local hypothermia and a powerful peripheral vasoconstriction during diving bouts are mechanisms by which penguins minimize heat losses and metabolic cost of thermoregulation (Dumonteil et al. 1994; Handrich et al. 1997), the overall daily field metabolic rate of penguins remains two to four times the shore resting metabolic rate at sea (Kooyman et al. 1982; Nagy et al. 2001; Froget et al. 2004). In line, experimental immersion into moderately cold water (8–10 °C) increases up to threefold the metabolic rate of penguins (Barré and Roussel 1986; Stahel and Nicol 1988; Dumonteil et al. 1994; Bevan et al. 1995; Fahlman et al. 2005). Therefore, the development of adaptive responses during the transition from shore to marine life represents a key step in the nutritional emancipation of penguin juveniles. Skeletal muscles are involved in locomotion and appear as the main source of regulatory thermogenesis in coldacclimated birds, producing metabolic heat by shivering and non-shivering mechanisms (Hissa 1988; Duchamp et al. 1999; Swanson and Vézina 2016). But, to be fully effective, muscle metabolism must be fuelled by commensurate energy substrate delivery. In birds, lipids are by far the main mobilized substrate for fuelling avian muscles at a high rate as it is the case during flight migration or cold acclimation (Dawson et al. 1983; Bénistant et al. 1998; Bedu et al. 2002; Vaillancourt et al. 2005; Weber 2009; Guglielmo 2010). Fatty acids also play a major role in regulatory thermogenesis by acting as stimulators of muscle thermogenic mechanisms in birds (Duchamp et al. 1999; Toyomizu et al. 2002; Talbot et al. 2004; Rey et al. 2010). Recently, we reported that lipids infusion in vivo induced a fourfold larger thermogenic effect in sea-acclimatized immature king penguins than in pre-fledging juveniles (Teulier et al. 2012). A high level of heat production that was associated with (1) a selective upregulation of lipid handling and oxidation in skeletal muscle (Teulier et al. 2012), and (2) a greater fatty acid-induced uncoupling of oxidative phosphorylation in muscle mitochondria (Talbot et al. 2004). However, the underlying energetic constraint responsible for the coordinated increases in lipid oxidation and thermogenic activity reported in sea-acclimatized penguins remains uncertain, as the effects of cold and exercise are difficult to discriminate. Indeed, cold acclimation leads to adaptive changes in skeletal muscles that resemble those resulting from endurance exercise, such as an increased muscle masses, capillary density and oxidative capacity and a shift toward slow-oxidative muscle fiber types (Duchamp et al. 1991, 1992; Mathieu-Costello et al. 1998; Ueda et al. 2005; Zhang et al. 2015). Furthermore, both
13
J Comp Physiol B
cold and exercise training trigger a coordinated increase in fatty acid supply to skeletal muscles (Dawson et al. 1983; Bénistant et al. 1998; Guglielmo 2010). The aim of this study was to disentangle the specific role of cold-water immersion from other aspects of sea acclimatization (swimming, diving) in the improvement of lipid catabolism and thermal ability. For this purpose, we simulated the passage to marine life by ten successive immersions in cold water in pre-fledging king penguin juveniles (Barré and Roussel 1986). We evaluated the capacity for adaptive thermogenesis by simultaneously measuring whole-body metabolic rate and shivering in penguins acutely exposed to different water temperatures. Finally, we evaluated the metabolic adjustments of lipid metabolism (1) in vivo by measuring the responses of metabolic rate and plasma metabolite concentrations to lipid infusion; and (2) in vitro by investigating muscle mitochondrial bioenergetics. Experimentally immersed penguins were compared to never-immersed pre-fledging juveniles.
Materials and methods Animals Field experiments were conducted on the Crozet archipelago (Possession Island, 46°25′ S, 51°45′ E) at the French Alfred Faure Station during six austral summer campaigns. According to the Agreed Measures for the Preservation of Antarctic and Sub-Antarctic Fauna, the project received the ethical approval of the Committee for the French Polar Research Institute (IPEV; program no. 131). King penguin juveniles (Aptenodytes patagonicus) of both sexes (12–14 months old) were captured on the nearby breeding colony of Baie du Marin before they had completed molting which is a pre-requisite for departing to sea. Captured birds finished their molt in an outside enclosure near the laboratory. Birds were weighed daily and force fed on Atlantic mackerel (Scomber vernalis). The amount of food (from 350 to 700 g per bird) was adjusted daily for each individual to maintain body mass. Half of the birds was fed for 3–5 days and then fasted for 48 h before experiments; they constituted the never-immersed (NI) group. In a second group, penguins were exposed to ten cold-water immersions at approximately 9–10 °C for 5 h per immersion every second day as previously described (Barré and Roussel 1986; Talbot et al. 2004). Noted that this water temperature was at approximately 5 °C warmer than temperatures usually undergone by king penguins in the austral Ocean (Charrassin and Bost 2001; Péron et al. 2012). Penguins were fed on a daily basis during the whole immersion protocol and then fasted for 48 h before experiments; they constituted the immersed (IM) group. On
J Comp Physiol B
completion of the study, all penguins were released at the site of their capture. Thermogenic activity in cold water Two successive austral summer campaigns (December– January 2005–2006 and 2006–2007) were devoted to simultaneously measure energy expenditure (indirect calorimetry) and shivering activity (electromyographic activity, EMG) of fully immersed penguin in vivo, at different water temperatures ranging from 29 to 12 °C. Twenty two birds were included in this protocol (12 NI and 10 IM weighing on average 8.2 ± 0.2 and 8.1 ± 0.2 kg, respectively). Penguins were placed in a thermostatic chamber consisting of an insulated 70-l polythene tank and left to equilibrate for 1 h at 10 °C in air before metabolic rate was monitored for 15 min. Then the tank was filled with water at 29 °C, the highest water temperature tested in the present protocol. To obtain metabolic steady state and thermal equilibrium, there was a 1 h adjustment period before the recording metabolic procedure was repeated. Thereafter, water temperature was reduced by steps, and after 1.5 h necessary to reach thermal equilibrium and metabolic steady state, the metabolic rate was measured over 15 min by indirect calorimetry using an open-circuit system (Barré and Roussel 1986; Teulier et al. 2012) at water temperatures ranging from 29 to 12 °C. Water temperature within the tank was monitored using a digital set-point potentiometric temperature controller coupled with a heater–cooler exchanger and a water-circulating pump. Body (stomach) temperature was continuously monitored during the experiment with a copper-constantan thermocouple. Shivering was measured as EMG activity of the pectoralis muscle. The EMG signal received from three monopolar electrodes insulated except for the tips (Stabilohm 110, Ni 80 % and Cr 20 %, 0.12 mm diameter) and acutely inserted into the muscle 10 mm apart was monitored on MP30B-CE, Biopac System and recorded with Biopac Student Lab Pro v.3.6.7 software (Santa Barbara, CA, USA) as previously described (Teulier et al. 2010). Catheterization and in vivo lipid oxidation One austral summer campaign (November–January 2008– 2009) was devoted to measure the metabolic effect of a triglyceride infusion in vivo. This protocol was performed at 10 °C (in air) within the thermoneutral zone for these birds (Froget et al. 2002). Eleven birds were included in this protocol (5 NI and 6 IM weighing on average 9.2 ± 0.2 and 8.0 ± 0.3 kg, respectively). Birds were catheterized and after stabilization within the thermostatic chamber for a night, metabolic rate was continuously recorded by indirect calorimetry following 30-min infusions at 1 mL/min
of saline (0.9 % NaCl) then Intralipid 20 % emulsion (Fresenius Kabi AB, Sweden) as described previously (Teulier et al. 2012). The day before experimentation, birds were equipped with intravenous catheters in marginal vein of each flipper as described previously (Teulier et al. 2012). Birds were thereafter placed in a thermostatic chamber at thermoneutrality during the night to ensure their complete recovery. The day of experiment, the first catheter (Braun Introcan 20G¼, 1.1 × 32 mm) was connected to a calibrated syringe pump (Kd Scientific, model 100, serial 931, USA) infusing continuously at 1 mL/min a saline solution (0.9 % NaCl) for 30 min. Eighty min after the end of saline infusion, a triglyceride emulsion (Intralipid® 20 %, Fresenius Kabi AB, Sweden) was infused during 30 min. The metabolic rate was continuously monitored (starting 45 min before the saline infusion and ending 2 h and a half after the end of triglyceride infusion) at 10 °C in air within the thermoneutral zone of these birds (Barré and Rouanet 1983). The in vivo total effect of triglyceride infusion upon metabolic rate was estimated by calculating the area under the curve (AUC) for each bird using trapezoidal integration minus the individual resting metabolic rate (Teulier et al. 2012). Blood sampling and kinetic parameters of metabolite disappearance The second catheter (BD Insyte-W 16G, 1.7 × 45 mm) was extended with 12 cm of Silastic® (Dow Corning, 0.64 × 1.19 mm, USA) to lessen the impact of vasoconstriction during blood sampling. Blood samples (500 µL) taken at different time points were centrifuged at 3500×g for 5 min at 4 °C and the resulting plasma samples stored at −80 °C until analysis of plasma triglyceride (TG), nonesterified fatty acid (NEFA), and glycerol concentrations using commercially available kits from Biomérieux (TG and glycerol) or Wako chemicals (NEFA). Kinetic parameters of plasma lipids were calculated using the first-order disappearance rate constant (k) obtained from linear regression of the logarithm of metabolite concentration against time data from the maximum metabolite concentration (Cmax) measured at the end of infusion period. The rate of disappearance (Rd) was calculated as (Rd = Cmax × k). In vitro mitochondrial respiration Two austral summer campaigns (December–January 2004–2005 and 2010–2011) were devoted to biochemical analysis of muscle mitochondria in vitro. Twenty birds were included in this protocol (14 NI and 6 IM weighing on average 7.8 ± 0.1 and 7.7 ± 0.2 kg, respectively). Superficial pectoralis muscle was surgically biopsied under general isoflurane anesthesia as described previously (Talbot
13
et al. 2004; Rey et al. 2008). The biopsy (1 g) was freshly used for mitochondria extraction and bioenergetics analysis. Intermyofibrillar and subsarcolemmal mitochondrial populations were isolated by a standard extraction protocol, involving potter homogenization, partial protease digestion and differential centrifugations (Talbot et al. 2004; Rey et al. 2008; Teulier et al. 2012). Mitochondria were pelleted at 8700×g and the amount of mitochondrial proteins was determined by a Biuret method with bovine serum albumin as standard. Mitochondrial oxygen consumption was measured using a Clark oxygen electrode (Rank Brothers LTD, Cambridge, UK) maintained at 38 °C and calibrated with an air-saturated respiratory medium [120 mM KCl, 5 mM KH2PO4, 1 mM MgCl2, 1 mM EGTA, 3 mM Hepes (pH 7.4) and 0.3 % (w/v) fatty acid-free bovine serum albumin]. Respiration was measured using NADH-linked substrates derived either from carbohydrate metabolism (5 mM pyruvate/2.5 mM malate) or lipid metabolism (40 µM palmitoyll-carnitine/2.5 mM malate), or a FADH2-linked substrate derived from citric acid cycle (5 mM succinate in the presence of 5 µM rotenone). The basal non-phosphorylating respiration rate (state 4oligo) was measured in the presence of 2.5 µg/mL oligomycin. The maximal fully uncoupled respiration rate (state FCCP) was initiated by the addition of 2 µM carbonyl cyanide p-tri-fluoro-methoxy-phenyl-hydrazone (FCCP). The respiratory control ratio (RCR) refers to the ratio of FCCP-induced maximal respiration rate (state FCCP) to basal non-phosphorylating respiration rate measured in the presence of oligomycin (state 4oligo). Mitochondrial oxidative phosphorylation efficiency One austral summer campaign (December–January 2013– 2014) was devoted to the biochemical analysis of muscle mitochondria in vitro. Fifteen birds were included in this protocol (8 NI and 7 IM weighing on average 9.3 ± 0.2 and 9.0 ± 0.3 kg, respectively). Superficial pectoralis muscle was surgically biopsied under general isoflurane anesthesia as described previously (Talbot et al. 2004; Rey et al. 2008). The biopsy (0.5 g) was freshly used for mitochondria extraction and bioenergetics analysis. Mitochondrial populations were isolated by a standard extraction protocol, involving potter homogenization, partial protease digestion and differential centrifugations (Monternier et al. 2014). The mitochondrial oxidative phosphorylation efficiency was assessed at 38 °C by measuring the rates of oxygen consumption and ATP synthesis in the respiratory medium supplemented with glucose (20 mM), hexokinase (1.5 U/ mL) and different concentrations of ADP ranging from 5 to 100 µM as described previously (Teulier et al. 2010; Monternier et al. 2014). Respiration was initiated by adding a mixture of respiratory substrates consisting of pyruvate (5 mM), malate (2.5 mM) and succinate (5 mM). Kinetics
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J Comp Physiol B
parameters of mitochondrial oxidation, i.e., the apparent affinity (Km) for ADP and the maximal oxidation rate (Vmax) of mitochondria, were determined from the dependence of mitochondrial oxygen consumption rate on ADP concentration ranging from 5 to 500 µM. Statistical analysis Relations between integrated EMG activity and water temperatures, or metabolic rate and water temperatures were expressed by two linear regression lines (Teulier et al. 2010) that intersect at the shivering threshold temperature (STT) or at the lower critical temperature (LCT), respectively. To draw these regression lines, we statistically determined by a paired-ANOVA test at which ambient temperature EMG activity or metabolic rate became significantly different from basal values, respectively. These values and those measured at lower water temperatures were then integrated in a second linear regression line distinct from the basal linear regression line. Two-way repeated-measure analyses of variances (RM ANOVA) followed by protected least significant difference tests were performed to estimate the effects of groups and infusions on metabolic rate and metabolite kinetics (SigmaPlot v.12, Systat Software, Inc., San Jose, CA, USA). When the assumptions of normality (Shapiro–Wilk test) or homoscedasticity (Levene test) were not met, Friedman RM ANOVA on ranks was used. Mitochondrial apparent Km for ADP and Vmax was determined for oxygen consumption by fitting experimental data by the Michaelis–Menten equation: V = [Vmax × (ADP)]/ [Km + (ADP)] using sigma plot 12.0 software. Two-way repeated-measure analyses of variances (RM ANOVA) followed by protected least significant difference tests were performed to estimate the effects of groups and ADP addition on mitochondrial respiratory rates (SigmaPlot v.12, Systat Software, Inc., San Jose, CA, USA). Mitochondrial oxidative phosphorylation and kinetic parameters were tested with analysis of variance (ANOVA) for independent values, followed by protected least significant difference tests (Statview v4.5 software). All data are presented as mean ± SEM with significance considered at p
