Dietary fatty acids and flight-training influence the expression of the eicosanoid hormone prostacyclin in songbirds.

Diet shifts can alter tissue fatty acid composition in birds, which is subsequently related to metabolic patterns. Eicosanoids, short-lived fatty acid-derived hormones, have been proposed to mediate these relationships but neither baseline concentrations nor the responses to diet and exercise have been measured in songbirds. We quantified a stable derivative of the vasodilatory eicosanoid prostacyclin in the plasma of male European Starlings (Sturnus vulgaris, N = 25) fed semisynthetic diets with either high (PUFA) or low (MUFA) amounts of n6 fatty acid precursors to prostacyclin. Plasma samples were taken from each bird before, immediately after, and two days following a 15-day flight-training regimen that a subset of birds (N = 17) underwent. We found elevated prostacyclin levels in flight-trained birds fed the PUFA diet compared to those fed the MUFA diet and a positive relationship between prostacyclin and body condition, indexed by fat score. Prostacyclin concentrations also significantly decreased at the final time point. These results are consistent with the proposed influences of precursor availability (i.e., dietary fatty acids) and regulatory feedback associated with exercise (i.e., fuel supply and inflammation), and suggest that prostacyclin may be an important mediator of dietary influence on songbird physiology.

Manipulation of photoperiod induces fat storage, but not fat mobilization in the migratory songbird, Dumetella carolinensis (Gray Catbird).

The annual cycle of migratory birds requires significant phenotypic remodeling. We sought to induce the migratory phenotype in Gray Catbirds by exposing them to a short-day light cycle. While adipose storage was stimulated, exceeding that typically seen in wild birds, other aspects of the migratory phenotype were unchanged. Of particular interest, the rate of lipid export from excised adipose tissue was nearly halved. This is in contrast to wild migratory birds in which lipid export rates are increased. These data suggest that exposure to an altered light cycle only activated the lipid storage program while inhibiting the lipid transport program. The factors governing lipid mobilization and transport remain to be elucidated.

Concerted phenotypic flexibility of avian erythrocyte size and number in response to dietary anthocyanin supplementation.

BACKGROUND: Endurance flight impose substantial oxidative costs on the avian oxygen delivery system. In particular, the accumulation of irreversible damage in red blood cells can reduce the capacity of blood to transport oxygen and limit aerobic performance. Many songbirds consume large amounts of anthocyanin-rich fruit, which is hypothesized to reduce oxidative costs, enhance post-flight regeneration, and enable greater aerobic capacity. While their antioxidant benefits appear most straightforward, the effects of anthocyanins on blood composition remain so far unknown. We fed thirty hand-raised European starlings (Sturnus vulgaris) two semisynthetic diets (with or without anthocyanin supplement) and manipulated the extent of flight activity in a wind tunnel (daily flying or non-flying for over two weeks) to test for their interactive effects on functionally important haematological variables. RESULTS: Supplemented birds had on average 15% more and 4% smaller red blood cells compared to non-supplemented individuals and these diet effects were independent of flight manipulation. Haemoglobin content was 7% higher in non-supplemented flying birds compared to non-flying birds, while similar haemoglobin content was observed among supplemented birds that were flown or not. Neither diet nor flight activity influenced haematocrit. CONCLUSION: The concerted adjustments suggest that supplementation generally improved antioxidant protection in blood, which could prevent the excess removal of cells from the bloodstream and may have several implications on the oxygen delivery system, including improved gas exchange and blood flow. The flexible haematological response to dietary anthocyanins may also suggest that free-ranging species preferentially consume anthocyanin-rich fruits for their natural blood doping, oxygen delivery-enhancement effects.

Flight and Dietary Antioxidants Influence Antioxidant Expression and Activity in a Migratory Bird.

Ecologically relevant factors such as exercise and diet quality can directly influence how physiological systems work including those involved in maintaining oxidative balance; however, to our knowledge, no studies to date have focused on how such factors directly affect expression of key components of the endogenous antioxidant system (i.e., transcription factors, select antioxidant genes, and corresponding antioxidant enzymes) in several metabolically active tissues of a migratory songbird. We conducted a three-factor experiment that tested the following hypotheses: (H1) Daily flying over several weeks increases the expression of transcription factors NRF2 and PPARs as well as endogenous antioxidant genes (i.e., CAT, SOD1, SOD2, GPX1, GPX4), and upregulates endogenous antioxidant enzyme activities (i.e., CAT, SOD, GPx). (H2) Songbirds fed diets composed of more 18:2n-6 PUFA are more susceptible to oxidative damage and thus upregulate their endogenous antioxidant system compared with when fed diets with less PUFA. (H3) Songbirds fed dietary anthocyanins gain additional antioxidant protection and thus upregulate their endogenous antioxidant system less compared with songbirds not fed anthocyanins. Flight training increased the expression of 3 of the 6 antioxidant genes and transcription factors measured in the liver, consistent with H1, but for only one gene (SOD2) in the pectoralis. Dietary fat quality had no effect on antioxidant pathways (H2), whereas dietary anthocyanins increased the expression of select antioxidant enzymes in the pectoralis, but not in the liver (H3). These tissue-specific differences in response to flying and dietary antioxidants are likely explained by functional differences between tissues as well as fundamental differences in their turnover rates. The consumption of dietary antioxidants along with regular flying enables birds during migration to stimulate the expression of genes involved in antioxidant protection likely through increasing the transcriptional activity of NRF2 and PPARs, and thereby demonstrates for the first time that these relevant ecological factors affect the regulation of key antioxidant pathways in wild birds. What remains to be demonstrated is how the extent of these ecological factors (i.e., intensity or duration of flight, amounts of dietary antioxidants) influences the regulation of these antioxidant pathways and thus oxidative balance.

Flight training and dietary antioxidants have mixed effects on the oxidative status of multiple tissues in a female migratory songbird.

Birds, like other vertebrates, rely on a robust antioxidant system to protect themselves against oxidative imbalance caused by energy-intensive activities such as flying. Such oxidative challenges may be especially acute for females during spring migration, as they must pay the oxidative costs of flight while preparing for reproduction; however, little previous work has examined how the antioxidant system of female spring migrants responds to dietary antioxidants and the oxidative challenges of regular flying. We fed two diets to female European starlings, one supplemented with a dietary antioxidant and one without, and then flew them daily in a windtunnel for 2 weeks during the autumn and spring migration periods. We measured the activity of enzymatic antioxidants (glutathione peroxidase, superoxide dismutase and catalase), non-enzymatic antioxidant capacity (ORAC) and markers of oxidative damage (protein carbonyls and lipid hydroperoxides) in four tissues: pectoralis, leg muscle, liver and heart. Dietary antioxidants affected enzymatic antioxidant activity and lipid damage in the heart, non-enzymatic antioxidant capacity in the pectoralis, and protein damage in leg muscle. In general, birds not fed the antioxidant supplement appeared to incur increased oxidative damage while upregulating non-enzymatic and enzymatic antioxidant activity, though these effects were strongly tissue specific. We also found trends for diet×training interactions for enzymatic antioxidant activity in the heart and leg muscle. Flight training may condition the antioxidant system of females to dynamically respond to oxidative challenges, and females during spring migration may shift antioxidant allocation to reduce oxidative damage.

Dietary linoleic acid, antioxidants, and flight training influence the activity of oxidative enzymes in European Starlings (Sturnus vulgaris).

Multiple studies have demonstrated that diet (e.g., fatty acid composition, antioxidants) and exercise training affect the metabolic performance of songbirds during aerobic activity, although the physiological mechanisms that cause such an effect remain unclear. We tested the hypothesis that elevated proportions of dietary linoleic acid (18:2n6) and amounts of dietary anthocyanins (a hydrophilic antioxidant class) influence the activity and protein expression of oxidative enzymes in flight and leg muscle of European Starlings (Sturnus vulgaris N = 96), a subset of which were flown over 15 days in a wind tunnel. Carnitine palmitoyl transferase (CPT) and citrate synthase (CS) activity displayed 18:2n6-dependent relationships with soluble protein concentration. Lactate dehydrogenase (LDH) was similarly related to protein concentration although also dependent on both dietary anthocyanins and flight training. 3-Hydroxyacyl CoA Dehydrogenase (HOAD) activity increased throughout the experiment in flight muscle, whereas this relationship was dependent on dietary anthocyanins in the leg muscle. Soluble protein concentration also increased throughout the experiment in the flight muscle, but was unrelated to date in the leg muscle, instead being influenced by both dietary anthocyanins and flight training. Training also produced additive increases in CPT and leg muscle HOAD activity. FAT/CD36 expression was related to both dietary 18:2n6 and training and changed over the course of the experiment. These results demonstrate a notable influence of our diet manipulations and flight training on the activity of these key oxidative enzymes, and particularly CPT and CS. Such influence suggests a plausible mechanism linking diet quality and metabolic performance in songbirds.

The energy savings-oxidative cost trade-off for migratory birds during endurance flight.

Elite human and animal athletes must acquire the fuels necessary for extreme feats, but also contend with the oxidative damage associated with peak metabolic performance. Here, we show that a migratory bird with fuel stores composed of more omega-6 polyunsaturated fats (PUFA) expended 11% less energy during long-duration (6 hr) flights with no change in oxidative costs; however, this short-term energy savings came at the long-term cost of higher oxidative damage in the omega-6 PUFA-fed birds. Given that fatty acids are primary fuels, key signaling molecules, the building blocks of cell membranes, and that oxidative damage has long-term consequences for health and ageing, the energy savings-oxidative cost trade-off demonstrated here may be fundamentally important for a wide diversity of organisms on earth.

Flight training in a migratory bird drives metabolic gene expression in the flight muscle but not liver, and dietary fat quality influences select genes.

Training and diet are hypothesized to directly stimulate key molecular pathways that mediate animal performance, and flight training, dietary fats, and dietary antioxidants are likely important in modulating molecular metabolism in migratory birds. This study experimentally investigated how long-distance flight training, as well as diet composition, affected the expression of key metabolic genes in the pectoralis muscle and the liver of European starlings (Sturnus vulgaris, n = 95). Starlings were fed diets composed of either a high or low polyunsaturated fatty acid (PUFA; 18:2n-6) and supplemented with or without a water-soluble antioxidant, and one-half of these birds were flight trained in a wind-tunnel while the rest were untrained. We measured the expression of 7 (liver) or 10 (pectoralis) key metabolic genes in flight-trained and untrained birds. Fifty percent of genes involved in mitochondrial metabolism and fat utilization were upregulated by flight training in the pectoralis (P < 0.05), whereas flight training increased the expression of only one gene responsible for fatty acid hydrolysis [lipoprotein lipase (LPL)] in the liver (P = 0.04). Dietary PUFA influenced the gene expression of LPL and fat transporter fatty acid translocase (CD36) in the pectoralis and one metabolic transcription factor [peroxisome proliferator-activated receptor (PPAR)-α (PPARα)] in the liver, whereas dietary antioxidants had no effect on the metabolic genes measured in this study. Flight training initiated a simpler causal network between PPARγ coactivators, PPARs, and metabolic genes involved in mitochondrial metabolism and fat storage in the pectoralis. Molecular metabolism is modulated by flight training and dietary fat quality in a migratory songbird, indicating that these environmental factors will affect the migratory performance of birds in the wild.

Dietary antioxidants attenuate the endocrine stress response during long-duration flight of a migratory bird.

Glucocorticoids (GCs) are metabolic hormones that promote catabolic processes, which release stored energy and support high metabolic demands such as during prolonged flights of migrating birds. Dietary antioxidants (e.g. anthocyanins) support metabolism by quenching excess reactive oxygen species produced during aerobic metabolism and also by activating specific metabolic pathways. For example, similar to GCs' function, anthocyanins promote the release of stored energy, although the extent of complementarity between GCs and dietary antioxidants is not well known. If anthocyanins complement GCs functions, birds consuming anthocyanin-rich food can be expected to limit the secretion of GCs when coping with a metabolically challenging activity, avoiding the exposure to potential hormonal detrimental effects. We tested this hypothesis in European starlings (Sturnus vulgaris) flying in a wind tunnel. We compared levels of corticosterone, the main avian GC, immediately after a sustained flight and at rest for birds that were fed diets with or without an anthocyanin supplement. As predicted, we found (i) higher corticosterone after flight than at rest in both diet groups and (ii) anthocyanin-supplemented birds had less elevated corticosterone after flight than unsupplemented control birds. This provides novel evidence that dietary antioxidants attenuate the activation of the HPA axis (i.e. increased secretion of corticosterone) during long-duration flight.

The effects of dietary linoleic acid and hydrophilic antioxidants on basal, peak, and sustained metabolism in flight-trained European starlings.

Dietary micronutrients have the ability to strongly influence animal physiology and ecology. For songbirds, dietary polyunsaturated fatty acids (PUFAs) and antioxidants are hypothesized to be particularly important micronutrients because of their influence on an individual's capacity for aerobic metabolism and recovery from extended bouts of exercise. However, the influence of specific fatty acids and hydrophilic antioxidants on whole-animal performance remains largely untested. We used diet manipulations to directly test the effects of dietary PUFA, specifically linoleic acid (18:2n6), and anthocyanins, a hydrophilic antioxidant, on basal metabolic rate (BMR), peak metabolic rate (PMR), and rates of fat catabolism, lean catabolism, and energy expenditure during sustained flight in a wind tunnel in European starlings (Sturnus vulgaris). BMR, PMR, energy expenditure, and fat metabolism decreased and lean catabolism increased over the course of the experiment in birds fed a high (32%) 18:2n6 diet, while birds fed a low (13%) 18:2n6 diet exhibited the reverse pattern. Additionally, energy expenditure, fat catabolism, and flight duration were all subject to diet-specific effects of whole-body fat content. Dietary antioxidants and diet-related differences in tissue fatty acid composition were not directly related to any measure of whole-animal performance. Together, these results suggest that the effect of dietary 18:2n6 on performance was most likely the result of the signaling properties of 18:2n6. This implies that dietary PUFA influence the energetic capabilities of songbirds and could strongly influence songbird ecology, given their availability in terrestrial systems.

PPAR expression, muscle size and metabolic rates across the gray catbird's annual cycle are greatest in preparation for fall migration.

Phenotypic flexibility across the annual cycle allows birds to adjust to fluctuating ecological demands. Varying energetic demands associated with time of year have been demonstrated to drive metabolic and muscle plasticity in birds, but it remains unclear what molecular mechanisms control this flexibility. We sampled gray catbirds at five stages across their annual cycle: tropical overwintering (January), northward spring (late) migration (early May), breeding (mid June), the fall pre-migratory period (early August) and southward fall (early) migration (end September). Across the catbird's annual cycle, cold-induced metabolic rate (V̇O2summit) was highest during migration and lowest during tropical wintering. Flight muscles exhibited significant hypertrophy and/or hyperplasia during fall migratory periods compared with breeding and the fall pre-migratory period. Changes in heart mass were driven by the tropical wintering stage, when heart mass was lowest. Mitochondrial content of the heart and pectoralis remained constant across the annual cycle as quantified by aerobic enzyme activities (CS, CCO), as did lipid catabolic capacity (HOAD). In the pectoralis, transcription factors PPARα, PPARδ and ERRß, coactivators PGC-1α and ß, and genes encoding proteins associated with fat uptake (FABPpm, Plin3) were unexpectedly upregulated in the tropical wintering stage, whereas those involved in fatty acid oxidation (ATGL, LPL, MCAD) were downregulated, suggesting a preference for fat storage over utilization. Transcription factors and coactivators were synchronously upregulated during pre-migration and fall migration periods in the pectoralis but not the heart, suggesting that these pathways are important in preparation for and during early migration to initiate changes to phenotypes that facilitate long-distance migration.

Conserved transcriptional activity and ligand responsiveness of avian PPARs: Potential role in regulating lipid metabolism in mirgratory birds.

Migratory birds undergo metabolic remodeling in tissues, including increased lipid storage in white adipose and fatty acid uptake and oxidation in skeletal muscle, to optimize energy substrate availability and utilization in preparation for long-distance flight. Different tissues undergo gene expression changes in keeping with their specialized functions and driven by tissue specific transcriptional pathways. Peroxisome proliferator-activated receptors (PPARs) are lipid-activated nuclear receptors that regulate metabolic pathways involved in lipid and glucose utilization or storage in mammals. To examine whether PPARs might mediate fatty acid activation of metabolic gene programs that would be relevant during pre-migratory fattening, we used gray catbird as the focal species. PPAR isoforms cloned from catbird share high amino acid identity with mammalian homologs (% vs human): gcPPARα (88.1%), gcPPARδ (87.3%), gcPPARγ (91.2%). We tested whether gcPPARs activated fatty acid (FA) utilization genes using Lpl and Cpt1b gene promoter-luciferase reporters in mammalian cell lines. In C2C12 mouse myocytes gcPPARα was broadly activated by the saturated and unsaturated FAs tested; while gcPPARδ showed highest activation by the mono-unsaturated FA, 18:1 oleic acid (+80%). In CV-1 monkey kidney cells gcPPARγ responded to the poly-unsaturated fatty acid, 20:5 eicosapentaenoic acid (+60%). Moreover, in agreement with their structural conservation, gcPPARs were activated by isoform selective synthetic agonists similar to the respective mammalian isoform. Adenoviral mediated over-expression of PPARα in C2C12 myocytes induced expression of genes involved in fatty acid transport, including Cd36/Fat, as well as Cpt1b, which mediates a key rate limiting step of mitochondrial ß-oxidation. These gene expression changes correlated with increased lipid droplet accumulation in C2C12 myoblasts and differentiated myotubes and enhanced ß-oxidation in myotubes. Collectively, the data predict that the PPARs play a conserved role in gray catbirds to regulate lipid metabolism in target tissues that undergo metabolic remodeling throughout the annual migratory cycle.

Environmental cues and dietary antioxidants affect breeding behavior and testosterone of male European starlings (Sturnus vulgaris).

Environmental cues, such as photoperiod, regulate the timing of major life-history events like breeding through direct neuroendocrine control. Less known is how supplementary environmental cues (e.g., nest sites, food availability) interact to influence key hormones and behaviors involved in reproduction, specifically in migratory species with gonadal recrudescence largely occurring at breeding sites. We investigated the behavioral and physiological responses of male European starlings to the sequential addition of nest boxes and nesting material, green herbs, and female conspecifics and how these responses depend on the availability of certain antioxidants (anthocyanins) in the diet. As expected, cloacal protuberance volume and plasma testosterone of males generally increased with photoperiod. More notably, testosterone levels peaked in males fed the high antioxidant diet when both nest box and herbal cues were present, while males fed the low antioxidant diet showed no or only a muted testosterone response to the sequential addition of these environmental cues; thus our results are in agreement with the oxidation handicap hypothesis. Males fed the high antioxidant diet maintained a constant frequency of breeding behaviors over time, whereas those fed the low antioxidant diet decreased breeding behaviors as environmental cues were sequentially added. Overall, sequential addition of the environmental cues modulated physiological and behavioral measures of reproductive condition, and dietary antioxidants were shown to be a key factor in affecting the degree of response to each of these cues. Our results highlight the importance of supplementary environmental cues and key resources such as dietary antioxidants in enhancing breeding condition of males, which conceivably aid in attraction of high quality females and reproductive success.

Annual life-stage regulation of lipid metabolism and storage and association with PPARs in a migrant species: the gray catbird (Dumetella carolinensis).

The annual cycle of a migrating bird involves metabolically distinct stages of substantial fatty acid storage and periods of increased fatty acid mobilization and utilization, and thus requires a great deal of phenotypic flexibility. Specific mechanisms directing stage transitions of lipid metabolism in migrants are largely unknown. This study characterized the role of the PPARs (peroxisome proliferator-activated receptors) in regulating migratory adiposity of the gray catbird (Dumetella carolinensis). Catbirds increased adipose storage during spring and autumn migration and showed increased rates of basal lipolysis during migration and tropical overwintering. Expression of the PPAR target genes involved in fat uptake and storage, FABPpm and PLIN3, increased during pre-migratory fattening. We found significant correlation between PPARγ and target gene expression in adipose but little evidence that PPARα expression levels drive metabolic regulation in liver during the migratory cycle.