In rainbow trout, dietary carbohydrates are poorly metabolized compared to other macronutrients. One prevalent hypothesis suggests that high dietary amino acid levels could contribute to the poor utilization of carbohydrates in trout. In mammals, alanine is considered an important gluconeogenic precursor, but has recently been found to stimulate AMP-activated protein kinase (AMPK) to reduce glucose levels. In trout, the effect of alanine on glucose fluxes is unknown. The goal of this study was to determine the effects of 4h exogenous alanine infusion on glucose metabolism in rainbow trout. Glucose fluxes, glucose appearance (Ra), and disposal (Rd) were measured in vivo. Key glycolytic and gluconeogenic enzyme expression and activity and cell signaling molecules relevant to glucose metabolism were assessed in liver and muscle. Results show that alanine inhibits Ra glucose (from 13.2+/-2.5 to 7.3+/-1.6 micromol / kg min) and Rd glucose (from 13.2+/-2.5 to 7.4+/-1.5 micromol / kg min) and the slight mismatch between Ra and Rd caused a reduction in glycemia, similar to the effects of insulin in trout. The reduction in Rd glucose can be partially explained by a reduction in glut4b expression in red muscle. In contrast to mammals, alanine-dependent glucose-lowering effects in trout did not involve hepatic AMPK activation, suggesting a different mechanistic basis. Interestingly, protein kinase B (AKT) activation increased only in muscle similar to effects observed in insulin-infused trout. We speculate that alanine-dependent effects were probably mediated through stimulation of insulin secretion which could indirectly promote alanine oxidation to provide the needed energy.

ABSTRACT Glucagon increases fish glycaemia, but how it affects glucose fluxes in vivo has never been characterized. The goal of this study was to test the hypothesis that glucagon stimulates hepatic glucose production (rate of appearance, R a ) and inhibits disposal (rate of disposal, R d ) in rainbow trout. Changes in the mRNA abundance of key proteins involved in glycolysis, gluconeogenesis and glycogen breakdown were also monitored. The results show that glucagon increases glycaemia (+38%) by causing a temporary mismatch between R a and R d before the two fluxes converge below baseline (−17%). A novel aspect of the regulation of trout gluconeogenesis is also demonstrated: the completely different effects of glucagon on the expression of three Pepck isoforms (stimulation of pck1 , inhibition of pck2a and no response of pck2b ). Glycogen phosphorylase was modulated differently among tissues, and muscle upregulated pygb and downregulated pygm . Glucagon failed to activate the cAMP-dependent protein kinase or FoxO1 signalling cascades. We conclude that trout hyperglycaemia results from the combination of two responses: (i) an increase in R a glucose induced by the stimulation of gluconeogenesis through transcriptional activation of pck1 (and possibly glycogen phosphorylase), and (ii) a decrease in R d glucose via inhibition of glycogen synthase and glycolysis. The observed decrease in glucose fluxes after 4 h of glucagon administration may be caused by a counter-regulatory response of insulin, potentially linked to the decrease in pygm transcript abundance. Overall, however, these integrated effects of glucagon only lead to modest changes in glucose fluxes that partly explain why trout seem to be unable to control glycaemia very tightly.

ABSTRACT The mobilization of mammalian lipid reserves is strongly stimulated during exercise to reach a maximum at moderate intensities, but the effects of swimming speed on fish lipolysis have never been quantified. Continuous infusion of 2-[ 3 H]glycerol was used to measure the rate of appearance of glycerol or lipolytic rate ( R a glycerol) in rainbow trout kept at rest, or during graded exercise in a swim tunnel up to critical swimming speed ( U crit ). Results show that R a glycerol is 1.67±0.18 µmol kg −1 min −1 in control animals, and remains at a steady level of 1.24±0.10 µmol kg −1 min −1 in exercising fish at all swimming intensities. Baseline lipolytic rate provides more than enough fatty acids from lipid reserves to accommodate all the oxidative fuel requirements for swimming at up to 2 body lengths per second (BL s −1 ), and more than 50% of the energy needed at U crit (3.4±0.1 BL s −1 ). Such ‘excess lipolysis’ also means that trout sustain high rates of fatty acid reesterification. Maintaining steady lipolysis at rest and throughout graded swimming is strikingly different from mammals that stimulate R a glycerol by twofold to fivefold to support exercise. Instead, trout act like ‘lipolytic machines’ that do not modulate R a glycerol even when their metabolic rate triples – a strategy that eliminates the need to increase lipolytic rate during exercise. This study also supports the notion that maintaining a high rate of reesterification (or triacylglycerol/fatty acid cycling) may be a mechanism widely used by ectotherms to achieve rapid membrane remodelling in variable environments.

ABSTRACT Muscle performance depends on the supply of metabolic fuels and disposal of end-products. Using circulating metabolite concentrations to infer changes in fluxes is highly unreliable because the relationship between these parameters varies greatly with physiological state. Quantifying fuel kinetics directly is therefore crucial to the understanding of muscle metabolism. This review focuses on how carbohydrates, lipids and amino acids are provided to fish muscles during hypoxia and swimming. Both stresses force white muscle to produce lactate at higher rates than it can be processed by aerobic tissues. However, lactate accumulation is minimized because disposal is also strongly stimulated. Exogenous supply shows that trout have a much higher capacity to metabolize lactate than observed during hypoxia or intense swimming. The low density of monocarboxylate transporters and their lack of upregulation with exercise explain the phenomenon of white muscle lactate retention. This tissue operates as a quasi-closed system, where glycogen stores act as an ‘energy spring’ that alternates between explosive power release during swimming and slow recoil from lactate in situ during recovery. To cope with exogenous glucose, trout can completely suppress hepatic production and boost glucose disposal. Without these responses, glycemia would increase four times faster and reach dangerous levels. The capacity of salmonids for glucoregulation is therefore much better than presently described in the literature. Instead of albumin-bound fatty acids, fish use lipoproteins to shuttle energy from adipose tissue to working muscles during prolonged exercise. Proteins may play an important role in fueling muscle work in fish, but their exact contribution is yet to be established. The membrane pacemaker theory of metabolism accurately predicts general properties of muscle membranes such as unsaturation, but it does not explain allometric patterns of specific fatty acids. Investigations of metabolic fuel kinetics carried out in fish to date have demonstrated that these ectotherms use several unique strategies to orchestrate energy supply to working muscles and to survive hypoxia.

ABSTRACT Rainbow trout are generally considered to be poor glucoregulators. To evaluate this, exogenous glucose was administered to chronically hyperglycemic fish at twice the endogenous rate of hepatic production, and their ability to modulate glucose fluxes was tested. Our goals were to determine: (1) whether hyperglycemic fish maintain higher glucose fluxes than normal; (2) whether they can lower hepatic production ( R a glucose) or stimulate disposal ( R d glucose) to cope with a carbohydrate overload; and (3) an estimate of the relative importance of glucose as an oxidative fuel. Results show that hyperglycemic trout sustain elevated baseline R a and R d glucose of 10.6±0.1 µmol kg −1 min −1 (or 30% above normal). If 50% of R d glucose was oxidized as in mammals, glucose could account for 36 to 100% of metabolic rate when exogenous glucose is supplied. In response to exogenous glucose, rainbow trout can completely suppress hepatic glucose production and increase disposal 2.6-fold, even with chronically elevated baseline fluxes. Such large changes in fluxes limit the increase in blood glucose to 2.5-fold and are probably mediated by the effects of insulin on glucose transporters 2 and 4 and on key enzymes of carbohydrate metabolism. Without this strong and rapid modulation of glucose kinetics, glycemia would rise four times faster to reach dangerous levels, exceeding 100 mmol l −1 . Such responses are typical of mammals, but rather unexpected for an ectotherm. The impressive plasticity of glucose kinetics demonstrated here suggests that trout have a much better glucoregulatory capacity than is usually portrayed in the literature.

ABSTRACT The ‘membrane pacemaker theory of metabolism’ proposes that membranes set metabolic rate by modulating protein activity, and thus purports to explain membrane fatty acid allometry. This relationship has never been tested across species in ectotherms. After accounting for phylogeny, recent analyses have failed to support this theory based on correlations between muscle membrane composition and body mass across mammals. Therefore, the goal of this study was to seek phylogenetically corrected correlations between membrane composition, body mass and calcium-ATPase activity, using 12 species of closely related cypriniform fish (4–5500 g) covering a much narrower genetic scale than in previous tests. The results show that fish membrane unsaturation decreases with mass, but through different mechanisms from those in endotherms: 16:0 replacing 22:6 in muscle and 18:0 replacing 16:1, 18:1 and 18:2 in liver. This shows that allometric patterns differ between endotherms and ectotherms as well as between tissues. After accounting for phylogeny, however, almost all these relationships lose significance except for overall unsaturation. No relationship between calcium-ATPase activity and mass or phospholipid composition was detected. This study shows that membrane unsaturation of cypriniforms decreases with mass, but that genetic cues unrelated to size account for differences in the relative abundance of individual fatty acids. The membrane pacemaker concept accurately predicts general membrane properties such as unsaturation, but fails to explain finer scale allometric patterns. Future examinations of the membrane pacemaker hypothesis will have to take into account that allometric patterns vary between endotherms and ectotherms and between tissues of the same animal class.

SUMMARY Changes in lactate kinetics as a function of exercise intensity have never been measured in an ectotherm. Continuous infusion of a tracer is necessary to quantify rates of lactate appearance ( R a ) and disposal ( R d ), but it requires double catheterization, which could interfere with swimming. Using rainbow trout, our goals were to: (1) determine the potential effects of catheters and blood sampling on metabolic rate ( Ṁ O 2 ), total cost of transport (TCOT), net cost of transport (NCOT) and critical swimming speed ( U crit ), and (2) monitor changes in lactate fluxes during prolonged, steady-state swimming or graded swimming from rest to U crit . This athletic species maintains high baseline lactate fluxes of 24 μmol kg −1 min −1 that are only increased at intensities >2.4 body lengths (BL) s −1 or 85% U crit . As the fish reaches U crit , R a is more strongly stimulated (+67% to 40.4 μmol kg −1 min −1 ) than R d (+41% to 34.7 μmol kg −1 min −1 ), causing a fourfold increase in blood lactate concentration. Without this stimulation of R d during intense swimming, lactate accumulation would double. By contrast, steady-state exercise at 1.7 BL s −1 increases lactate fluxes to ~30 μmol kg −1 min −1 , with a trivial mismatch between R a and R d that only affects blood concentration minimally. Results also show that the catheterizations and blood sampling needed to measure metabolite kinetics in exercising fish have no significant impact on Ṁ O 2 or TCOT. However, these experimental procedures affect locomotion energetics by increasing NCOT at high speeds and by decreasing U crit .

SUMMARY The effects of short-term food deprivation on lipid metabolism are well documented, but little is known about prolonged fasting. This study monitored the kinetics of glycerol (rate of appearance, R a glycerol) and non-esterified fatty acids (R a NEFA) in fasting rabbits. Our goals were to determine whether lipolysis is stimulated beyond values seen for short-term fasting, and to characterize the roles of primary (intracellular) and secondary (with transit through the circulation) triacylglycerol/fatty acid cycling (TAG/FA cycling) in regulating fatty acid allocation to oxidation or re-esterification. R a glycerol (9.62±0.72 to 15.29±0.96 μmol kg –1 min –1 ) and R a NEFA (18.05±2.55 to 31.25±1.93 μmol kg –1 min –1 ) were stimulated during the first 2 days of fasting, but returned to baseline after 4 days. An initial increase in TAG/FA cycling was followed by a reduction below baseline after 6 days without food, with primary and secondary cycling contributing to these responses. We conclude that the classic activation of lipolysis caused by short-term fasting is abolished when food deprivation is prolonged. High rates of re-esterification may become impossible to sustain, and TAG/FA cycling could decrease to reduce its cost to 3% of total energy expenditure. Throughout prolonged fasting, fatty acid metabolism gradually shifts towards increased oxidation and reduced re-esterification. Survival is achieved by pressing fuel selection towards the fatty acid dominance of energy metabolism and by slowing substrate cycles to assist metabolic suppression. However, TAG/FA cycling remains active even after prolonged fasting, suggesting that re-esterification is a crucial mechanism that cannot be stopped without harmful consequences.

Summary Animals must regulate the fluxes of multiple fuels to support changing metabolic rates that result from variation in physiological circumstances. The aim of fuel selection strategies is to exploit the advantages of individual substrates while minimizing the impact of disadvantages. All exercising mammals share a general pattern of fuel selection: at the same they oxidize the same ratio of lipids to carbohydrates. However, highly aerobic species rely more on intramuscular fuels because energy supply from the circulation is constrained by trans-sarcolemmal transfer. Fuel selection is performed by recruiting different muscles, different fibers within the same muscles or different pathways within the same fibers. Electromyographic analyses show that shivering humans can modulate carbohydrate oxidation either through the selective recruitment of type II fibers within the same muscles or by regulating pathway recruitment within type I fibers. The selection patterns of shivering and exercise are different: at the same , a muscle producing only heat (shivering) or significant movement (exercise) strikes a different balance between lipid and carbohydrate oxidation. Long-distance migrants provide an excellent model to characterize how to increase maximal substrate fluxes. High lipid fluxes are achieved through the coordinated upregulation of mobilization, transport and oxidation by activating enzymes, lipid-solubilizing proteins and membrane transporters. These endurance athletes support record lipolytic rates in adipocytes, use lipoprotein shuttles to accelerate transport and show increased capacity for lipid oxidation in muscle mitochondria. Some migrant birds use dietary omega-3 fatty acids as performance-enhancing agents to boost their ability to process lipids. These dietary fatty acids become incorporated in membrane phospholipids and bind to peroxisome proliferator-activated receptors to activate membrane proteins and modify gene expression. Summary Animals must regulate the fluxes of multiple fuels to support changing metabolic rates that result from variation in physiological circumstances. The aim of fuel selection strategies is to exploit the advantages of individual substrates while minimizing the impact of disadvantages. All exercising mammals share a general pattern of fuel selection: at the same they oxidize the same ratio of lipids to carbohydrates. However, highly aerobic species rely more on intramuscular fuels because energy supply from the circulation is constrained by trans-sarcolemmal transfer. Fuel selection is performed by recruiting different muscles, different fibers within the same muscles or different pathways within the same fibers. Electromyographic analyses show that shivering humans can modulate carbohydrate oxidation either through the selective recruitment of type II fibers within the same muscles or by regulating pathway recruitment within type I fibers. The selection patterns of shivering and exercise are different: at the same , a muscle producing only heat (shivering) or significant movement (exercise) strikes a different balance between lipid and carbohydrate oxidation. Long-distance migrants provide an excellent model to characterize how to increase maximal substrate fluxes. High lipid fluxes are achieved through the coordinated upregulation of mobilization, transport and oxidation by activating enzymes, lipid-solubilizing proteins and membrane transporters. These endurance athletes support record lipolytic rates in adipocytes, use lipoprotein shuttles to accelerate transport and show increased capacity for lipid oxidation in muscle mitochondria. Some migrant birds use dietary omega-3 fatty acids as performance-enhancing agents to boost their ability to process lipids. These dietary fatty acids become incorporated in membrane phospholipids and bind to peroxisome proliferator-activated receptors to activate membrane proteins and modify gene expression.

SUMMARY Current understanding of lactate metabolism in fish is based almost entirely on the interpretation of concentration measurements that cannot be used to infer changes in flux. The goals of this investigation were: (1) to quantify baseline lactate fluxes in rainbow trout ( Oncorhynchus mykiss ) under normoxic conditions; (2) to establish how changes in rates of lactate appearance ( R a ) and disposal ( R d ) account for the increase in blood lactate elicited by hypoxia; and (3) to identify the tissues responsible for lactate production. R a and R d lactate of rainbow trout were measured in vivo by continuous infusion of [U- 14 C]lactate in trout exposed to 25% O 2 saturation or maintained in normoxia for 90 min. In normoxic fish, R a lactate decreased from 18.2 to 13.1 μmol kg –1 min –1 and R d lactate from 19.0 to 12.8. R a and R d were always matched, thereby maintaining a steady baseline blood lactate concentration of ∼0.8 mmol l –1 . By contrast, the hypoxic fish increased blood lactate to 8.9 mmol l –1 and R a lactate from 18.4 to 36.5 μmol kg –1 min –1 . This stimulation of anaerobic glycolysis was unexpectedly accompanied by a 52% increase in R d lactate from 19.9 to 30.3 μmol kg –1 min –1 . White muscle was the main producer of lactate, which accumulated to 19.2 μmol g –1 in this tissue. This first study of non-steady-state lactate kinetics in fish shows that the increase in lactate disposal elicited by hypoxia plays a strategic role in reducing the lactate load on the circulation. Without this crucial response, blood lactate accumulation would double.

SUMMARY Wild semipalmated sandpipers ( Calidris pusilla ) eat n-3 fatty acids to prime their muscles for long migrations. Sedentary bobwhite quails( Colinus virginianus ) were used as a model to investigate the mechanisms for this natural doping. Our goal was to characterize the stimulating effects of n-3 eicosapentaenoic acid (EPA) and n-3 docosahexaenoic acid (DHA) on oxidative capacity. Mechanisms linked to changes in membrane composition and in gene expression for peroxisome proliferator-activated receptors (PPAR) were investigated. Dietary n-3 fatty acids stimulated the activities of oxidative enzymes by 58–90% (citrate synthase, cytochrome oxidase, carnitine palmitoyl transferase and hydroxyacyl dehydrogenase), and sedentary quails showed the same changes in membrane composition as sandpipers preparing for migration. EPA and DHA have the same doping effect. The substitution of n-6 arachidonic acid by n-3 EPA in membrane phospholipids plays an important role in mediating the metabolic effects of the diet, but results provide no significant support for the involvement of PPARs (as determined by changes in gene expression). The fatty acid composition of mitochondrial membranes and sarcoplasmic reticulum can be monitored by measuring total muscle phospholipids because all phospholipids are equally affected by diet. Only extreme regimes of endurance training can lead to increments in oxidative capacity matching those induced here by diet. As they prepare for long migrations, semipalmated sandpipers improve their physical fitness by eating! Choosing n-3 fatty acid doping over endurance training strikes us as a better strategy to boost aerobic capacity when rapid storage of energy is critical.

SUMMARY Long-distance migrants have evolved specific adaptations that make their athletic records possible. Unique mechanisms explaining their amazing capacity for endurance exercise have now been uncovered, particularly with respect to energy storage, mobilization, transport and utilization. Birds are champions of migration because flying offers a key compromise: it allows more rapid movement than swimming, but has a lower cost of transport than running. High efficiency for muscle contraction, pointed wings, low wingloading, travelling in V-formations, storing fuel as energy-dense lipids and atrophy of non-essential organs are some of their strategies to decrease the cost of transport. The ability to process lipids rapidly also emerges as a crucial component of the migrant phenotype. High lipid fluxes are made possible by lipoprotein shuttles and fatty acid binding proteins (FABPs) that accelerate lipid transport and by upgrading the metabolic machinery for lipolysis and lipid oxidation. Preparation for long flights can include natural doping on n-3 polyunsaturated fatty acids (n-3 PUFAs) from unique invertebrate diets. Muscle performance is improved by restructuring membrane phospholipids and by activating key genes of lipid metabolism through peroxisome proliferator-activated receptors (PPARs). The physiological secret to long migrations does not depend on a single `magic' adaptation but on the integration of multiple adjustments in morphology, biomechanics, behavior,nutrition and metabolism. Research on the physiology of migrants improves the fundamental knowledge of exercise biology, but it also has important implications for wildlife conservation, treating obesity and improving the performance of human athletes.

SUMMARY Lipolysis provides fatty acids that support key life processes by functioning as membrane components, oxidative fuels and metabolic signals. It is commonly measured as the rate of appearance of glycerol( R a glycerol). Its in vivo regulation by catecholamines has been thoroughly investigated in mammals, but little information is available for ectotherms. Therefore, the goals of this study were, first, to characterize the effects of the catecholamines norepinephrine(NE) and epinephrine (Epi) on the lipolytic rate of intact rainbow trout( Oncorhynchus mykiss ) and, second, to determine whether the plasma glycerol concentration is a reliable index of R a glycerol. Our results show that baseline R a glycerol (4.6±0.4μmol kg –1 min –1 ) is inhibited by NE(–56%), instead of being stimulated, as in mammals, whereas Epi has the same activating effect in both groups of vertebrates (+167%). NE-induced inhibition of fish lipolysis might play a particularly important role during aquatic hypoxia, when survival often depends on regulated metabolic depression. The plasma glycerol concentration is a poor predictor of R a glycerol, and it should not be used as an index of lipolysis. Trout maintain a particularly high baseline lipolytic rate because only 13% of the fatty acids provided are sufficient to support total energy expenditure, whereas the remaining fatty acids must undergo reesterification(87%).

SUMMARY Fish endurance swimming is primarily powered by lipids supplied to red muscle by the circulation, but the mechanism of delivery remains unknown. By analogy to mammals, previous studies have focused on non-esterified fatty acids (NEFA bound to albumin), but lipoproteins have not been considered as an energy shuttle to working muscles. The effects of exercise on fish lipoprotein lipase (LPL) have never been investigated. We hypothesized that LPL and circulating lipoproteins would be modified by prolonged swimming. Because LPL is naturally bound to the endothelium, we have used heparin to release the enzyme in the circulation and to characterize reserve capacity for lipoprotein catabolism. The effects of exercise (4 days at 1.5 body lengths s –1 in a swim tunnel) were measured for red muscle LPL,post-heparin plasma LPL, and lipoprotein concentration/composition. Red muscle LPL activity increased from 18±5 (rest) to 49± 9 nmol fatty acids min –1 g –1 (swimming). In resting fish,heparin administration caused a 27-fold increase in plasma LPL activity that reached a maximum of 1.32± 0.67 μmol fatty acids min –1 ml –1 plasma. This heparin-induced response of plasma LPL was not different between resting controls and exercised fish. Heparin or prolonged swimming had no effect on the concentration/composition of lipoproteins that contain 92% of the energy in total plasma lipids. We conclude that (1) red muscle LPL is strongly activated by endurance swimming, (2) rainbow trout have a high reserve capacity for hydrolyzing lipoproteins, and (3) future studies should aim to measure lipoprotein flux because their concentration does not reflect changes in flux. These novel characteristics of fish LPL imply that lipoproteins are used as a metabolic shuttle between fat reserves and working muscles, a strategy exploiting an abundant source of energy in rainbow trout.

For long migrations, birds must rely on high flux capacities at all steps of lipid metabolism, from the mobilization of adipose reserves to fatty acid oxidation in flight muscle mitochondria. Substrate kinetics and indirect calorimetry were used to investigate key parameters of lipid metabolism in a highly aerobic shorebird: the ruff sandpiper Philomachus pugnax . In this study, we have quantified the effects of cold exposure because such measurements are presently impossible during flight. Lipolytic rate was monitored by continuous infusion of 2-[ 3 H]-glycerol and lipid oxidation by respirometry. Plasma lipid concentrations (non-esterified fatty acids, neutral lipids and phospholipids) and their fatty acid composition were also measured to assess whether cold exposure causes selective metabolism of specific lipids. Results show that shivering leads to a 47% increase in metabolic rate (44.4±3.8 ml O 2 kg –1 min –1 to 65.2±8.1 ml O 2 kg –1 min –1 ), almost solely by stimulating lipid oxidation (33.3± 3.3 ml O 2 kg –1 min –1 to 48.2±6.8 ml O 2 kg –1 min –1 ) because carbohydrate oxidation remains close to 11.5± 0.5 ml O 2 kg –1 min –1 . Sandpipers support an unusually high lipolytic rate of 55–60 μmol glycerol kg –1 min –1 . Its stimulation above thermoneutral rates is unnecessary during shivering when the birds are still able to re-esterify 50% of released fatty acids. No changes in plasma lipid composition were observed, suggesting that cold exposure does not lead to selective metabolism of particular fatty acids. This study provides the first measurements of lipolytic rate in migrant birds and shows that their capacity for lipid mobilization reaches the highest values measured to date in vertebrates. Extending the limits of conventional lipid metabolism has clearly been necessary to achieve long-distance migrations.

SUMMARY During their fall migration from the Arctic to South America, semipalmated sandpipers Calidris pusilla stop in the Bay of Fundy (east coast of Canada) before flying non-stop for ∼4500 km across the ocean. Refueling birds double their body mass by feeding on Corophium volutator , an amphipod containing high amounts of n-3 polyunsaturated fatty acids (n-3 PUFA), particularly eicosapentaenoic (20:5) and docosahexaenoic acid (22:6). In mammals, high dietary intake of n-3 PUFA is known to increase capacity for oxidative metabolism. Therefore, we hypothesized that tissue incorporation of n-3 PUFA would be associated with increases in the activity of key muscle enzymes to upregulate energy metabolism for prolonged exercise. Birds were collected at various stages of fat loading to monitor changes in lipid composition and flight muscle enzymes simultaneously. Enzymes were measured to assess oxidative capacity [citrate synthase (CS)],β-oxidation [carnitine palmitoyl transferase (CPT) and 3-hydroxyacyl dehydrogenase (HOAD)] and glycolytic capacity [lactate dehydrogenase (LDH)]. Changes in the fatty acid composition of muscle membranes (phospholipids) and fuel reserves (neutral lipids) were measured separately to distinguish between membrane-related and systemic effects of n-3 PUFA. Results show that muscle CS and HOAD are stimulated during refueling and that their activities are correlated with n-3 PUFA content in phospholipids (22:6 for CS, 20:5 for HOAD)and in neutral lipids (20:5 for CS). This suggests that 20:5 and 22:6 have different effects on energy metabolism and that they act via changes in membrane structure and systemic mechanisms. CPT and LDH did not change during refueling, but LDH activity was significantly related to the n-3 PUFA content of fuel reserves. This study shows that oxidative capacity increases rapidly during refueling and supports the idea that dietary n-3 PUFA are used as molecular signals to prime flight muscles of some long-distance migrants for extreme exercise.

SUMMARY At the end of summer, semipalmated sandpipers ( Calidris pusilla )traveling from the Arctic stop in the Bay of Fundy (east coast of Canada) to build large fat reserves before a non-stop flight to South America. During a 2-week stopover, the body mass of this small shorebird is doubled (∼20 g to 40 g) by feeding on a burrowing amphipod, Corophium volutator ,that contains unusually high levels of n-3 polyunsaturated fatty acids (PUFA). In mammals, high n-3 PUFA content of membrane phospholipids (PL) is linked to improved exercise performance due to increased membrane fluidity that accelerates transmembrane lipid transport. We hypothesized that dietary n-3 PUFA could be used as a natural `performance-enhancing substance' by semipalmated sandpipers to prepare their flight muscles for migration. Also,PUFA stored as fuel in neutral lipids (NL) can be mobilized more quickly than saturated fatty acids, but they contain less energy per unit mass. It is therefore unclear whether dietary fatty acids are modified before storage. Birds were collected at various stages of fat loading to examine changes in the composition of tissue PL (membranes) and NL (fuel stores). Results show that dietary n-3 PUFA are incorporated in tissue lipids in less than 2 weeks. During the stopover, the double bond index of muscle PL increases by 25% and the fatty acid profiles of both muscle PL and adipose NL converge with that of the diet. However, >50% of dietary n-3 PUFA are converted to other fatty acids before storage, mainly to oleate (18:1), possibly because monounsaturates offer a compromise between high energy density and ease of mobilization. This study shows that long-distance migrant birds can (1) use natural diets rich in specific lipids to prime flight muscles for endurance exercise, and (2) modify dietary fatty acids before storing them as fuel.

SUMMARY The metabolic consequences of cold exposure and exercise are not well characterized in birds. Ruff sandpipers Philomachus pugnax are migrant shorebirds traveling between Africa and Siberia for up to 30 000 km annually. Our goal was to quantify the fuel selection pattern of these remarkable athletes during shivering and terrestrial locomotion. We used indirect calorimetry and nitrogen excretion analysis to measure their rates of lipid, carbohydrate and protein oxidation at different temperatures (22, 15,10 or 5°C) and different treadmill speeds (15, 20, 25, 30, 35 or 40 m min –1 ). Results show that lipid oxidation supplies nearly all the energy necessary to support shivering and running, and that the pattern of oxidative fuel selection is independent of shivering or running intensity. During shivering, total ATP production is unequally shared between lipids(82%), carbohydrates (12%) and proteins (6%). During running, lipids remain the dominant substrate (66%), with carbohydrates (29%) and proteins (5%)playing more minor roles. The prevailing use of lipids during intense shivering and high-speed running is not consistent with the fuel selection pattern observed in exercising and cold-exposed mammals. The exact mechanisms allowing birds to use lipids at extremely high rates are still largely unexplored, and quantifying the relative importance of different fuels during long-distance flight remains a major challenge for future research.

SUMMARY The energy metabolism of hibernators has not been characterized for normothermic fasting, and our goal was to quantify oxidative fuel selection of non-hibernating woodchucks Marmota monax during prolonged food deprivation. Indirect calorimetry and nitrogen excretion measurements were used to assess changes in metabolic rate( V̇ O 2 ), fuel selection and composition of nitrogen wastes, as well as seasonal differences. For reference, matching experiments were also performed on rabbits. The results show that woodchucks have a higher metabolic rate in summer (271μmol O 2 kg -1 min -1 ) than in spring (200μmol O 2 kg -1 min -1 ) and that fasting-induced metabolic depression is only possible in summer (-25% in 14 days). The metabolic rate of rabbits is high at all times (383 μmol O 2 kg -1 min -1 ), but they show a more rapid depression in response to fasting (-32% in 7 days). Woodchucks have a naturally low reliance on proteins in the fed state (accounting for 8% V̇ O 2 in spring;17% V̇ O 2 in summer; vs 28% V̇ O 2 in rabbits)and are able to decrease it even further during fasting (spring, 5% V̇ O 2 ; summer, 6% V̇ O 2 ; vs 20% V̇ O 2 in rabbits). This study shows that, apart from their notorious capacity for hibernation, woodchucks are particularly well adapted for normothermic fasting. Their ability to cope with prolonged food deprivation is based on a series of integrated responses eliciting deep metabolic depression and a rapid change in fuel selection to spare limited protein reserves. Information presently available on prolonged fasting suggests that such an ability for metabolic depression, possibly down to minimal levels still compatible with normothermic life, may be common among mammals. In contrast, the extreme protein sparing demonstrated in woodchucks is a unique metabolic feature of fasting champions.