Comprehensive endocrine response to acute stress in the bottlenose dolphin from serum, blubber, and feces.

Several hormones are potential indicators of stress in free-ranging animals and provide information on animal health in managed-care settings. In response to stress, glucocorticoids (GC, e.g. cortisol) first appear in circulation but are later incorporated into other tissues (e.g. adipose) or excreted in feces or urine. These alternative matrices can be sampled remotely, or by less invasive means, than required for blood collection and are especially valuable in highly mobile species, like marine mammals. We characterized the timing and magnitude of several hormones in response to a stressor in bottlenose dolphins (Tursiops truncatus) and the subsequent incorporation of cortisol into blubber, and its metabolites excreted in feces. We evaluated the endocrine response to an acute stressor in bottlenose dolphins under managed care. We used a standardized stress protocol where dolphins voluntarily beached onto a padded platform and remained out of water for two hours; during the stress test blood samples were collected every 15 min and blubber biopsies were collected every hour (0, 60, and 120 min). Each subject was studied over five days: voluntary blood samples were collected on each of two days prior to the stress test; 1 and 2 h after the conclusion of the out-of-water stress test; and on the following two days after the stress test. Fecal samples were collected daily, each afternoon. The acute stressor resulted in increases in circulating ACTH, cortisol, and aldosterone during the stress test, and each returned to baseline levels within 2 h of the dolphin's return to water. Both cortisol and aldosterone concentrations were correlated with ACTH, suggesting both corticosteroids are at least partly regulated by ACTH. Thyroid hormone concentrations were generally unaffected by the acute stressor. Blubber cortisol increased during the stress test, and fecal GC excretion was elevated on the day of the stress test. We found that GCs in bottlenose dolphins can recover within hours of acute stress, and that cortisol release can be detected in alternate matrices within a few hours-within 2 h in blubber, and 3.5-5 h in fecal samples.

The demands of lactation promote differential regulation of lipid stores in fasting elephant seals.

Fasting animals must ration stored reserves appropriately for metabolic demands. Animals that experience fasting concomitant with other metabolically demanding activities are presented with conflicting demands of energy conservation and expenditure. Our objective was to understand how fasting northern elephant seals regulate the mobilization of lipid reserves and subsequently milk lipid content during lactation. We sampled 36 females early and 39 at the end of lactation. To determine the separate influences of lactation from fasting, we also sampled fasting but non-lactating females early and late (8 and 6 seals, respectively) in their molting fasting period. Mass and adiposity were measured, as well as circulating non-esterified fatty acid (NEFA), triacylglycerol (TAG), cortisol, insulin and growth hormone levels. Milk was collected from lactating females. Milk lipid content increased from 31% in early to 51% in late lactation. In lactating females plasma NEFA was positively related to cortisol and negatively related to insulin, but in molting seals, only variation in cortisol was related to NEFA. Milk lipid content varied with mass, adiposity, NEFA, TAG, cortisol and insulin. Surprisingly, growth hormone concentration was not related to lipid metabolites or milk lipid. Suppression of insulin release appears to be the differential regulator of lipolysis in lactating versus molting seals, facilitating mobilization of stored lipids and maintenance of high NEFA concentrations for milk synthesis. Milk lipid was strongly impacted by the supply of substrate to the mammary gland, indicating regulation at the level of mobilization of lipid reserves.

Natural Variation in Stress Hormones, Comparisons Across Matrices, and Impacts Resulting from Induced Stress in the Bottlenose Dolphin.

Knowledge regarding stress hormones and how they vary in response to seasonality, gender, age, and reproductive status for any marine mammal is limited. Furthermore, stress hormones may be measured in more than one matrix (e.g., feces, blood, blubber), but the relationships between levels of a given hormone across these matrices are unknown, further complicating the interpretations of hormones measured in samples collected from wild animals. A study is underway to address these issues in a population of bottlenose dolphins trained for voluntary participation in sample collections from different matrices and across season and time of day.

Oxidative stress in northern elephant seals: Integration of omics approaches with ecological and experimental studies.

Northern elephant seals experience conditions that increase oxidative stress (OS), including extended fasting, ischemia and hypoxia during breath-holds, and immune responses during colonial breeding. Increased OS is suggested by increases in tissue and plasma concentrations of pro-oxidant enzymes NADPH oxidase and xanthine oxidase (XO). Serum cortisol concentrations were positively associated with XO concentrations and damage markers. Elephant seals exhibit robust anti-oxidant responses as evidenced by increases in anti-oxidant enzymes in plasma and tissues. These responses were sufficient to prevent oxidative damage during breath-holds and extended fasts in juveniles. However, high rates of energy expenditure during breeding were associated with increased evidence for oxidative damage to lipids, proteins and DNA in adults. We integrated investigations of the fasting metabolome and muscle and blubber transcriptomes into our oxidative stress studies. Non-targeted metabolomics analysis of fasting seals identified 227 known metabolites in plasma, including those related to glutathione and purine metabolism. Changes in plasma metabolites suggested that glutathione biosynthesis increased during fasting in weaned pups but not in lactating females. We produced the first reference sequence for elephant seals by RNA sequencing of skeletal muscle and adipose tissue transcriptomes and de novo transcriptome assembly. We annotated muscle and adipose transcripts and identified thousands of genes, including potential mediators of OS. This resource provides elephant seal-specific gene sequences, complements existing metabolite and protein expression studies and provides tools for examining cellular responses to OS in a variety of contexts. We examined changes in tissue gene expression in response to experimental elevation of plasma cortisol. Responses included downregulation of Negative Regulator of Reactive Oxygen Species (NRROS) in muscle, a regulator that limits reactive oxygen species production by tissues. These tools provide novel views of the cellular and systemic mechanisms that enable seals to tolerate high levels of OS.

Muscle transcriptome response to ACTH administration in a free-ranging marine mammal.

While much of our understanding of stress physiology is derived from biomedical studies, little is known about the downstream molecular consequences of adaptive stress responses in free-living animals. We examined molecular effectors of the stress hormones cortisol and aldosterone in the northern elephant seal, a free-ranging study system in which extreme physiological challenges and cortisol fluctuations are a routine part of life history. We stimulated the neuroendocrine stress axis by administering exogenous adrenocorticotropic hormone (ACTH) and examined the resultant effects by measuring corticosteroid hormones, metabolites, and gene expression before, during, and following administration. ACTH induced an elevation in cortisol, aldosterone, glucose, and fatty acids within 2 h, with complete recovery observed within 24 h of administration. The global transcriptional response of elephant seal muscle tissue to ACTH was evaluated by transcriptomics and involved upregulation of a highly coordinated network of conserved glucocorticoid (GC) target genes predicted to promote metabolic substrate availability without causing deleterious effects seen in laboratory animals. Transcriptional recovery from ACTH was characterized by downregulation of GC target genes and restoration of cell proliferation, metabolism, and tissue maintenance pathways within 24 h. Differentially expressed genes included several adipokines not previously described in muscle, reflecting unique metabolic physiology in fasting-adapted animals. This study represents one of the first transcriptome analyses of cellular responses to hypothalamic-pituitary-adrenal axis stimulation in a free-living marine mammal and suggests that compensatory, tissue-sparing mechanisms may enable marine mammals to maintain cortisol and aldosterone sensitivity while avoiding deleterious long-term consequences of stress.

Transcriptome analysis of northern elephant seal (Mirounga angustirostris) muscle tissue provides a novel molecular resource and physiological insights.

BACKGROUND: The northern elephant seal, Mirounga angustirostris, is a valuable animal model of fasting adaptation and hypoxic stress tolerance. However, no reference sequence is currently available for this and many other marine mammal study systems, hindering molecular understanding of marine adaptations and unique physiology. RESULTS: We sequenced a transcriptome of M. angustirostris derived from muscle sampled during an acute stress challenge experiment to identify species-specific markers of stress axis activation and recovery. De novo assembly generated 164,966 contigs and a total of 522,699 transcripts, of which 68.70% were annotated using mouse, human, and domestic dog reference protein sequences. To reduce transcript redundancy, we removed highly similar isoforms in large gene families and produced a filtered assembly containing 336,657 transcripts. We found that a large number of annotated genes are associated with metabolic signaling, immune and stress responses, and muscle function. Preliminary differential expression analysis suggests a limited transcriptional response to acute stress involving alterations in metabolic and immune pathways and muscle tissue maintenance, potentially driven by early response transcription factors such as Cebpd. CONCLUSIONS: We present the first reference sequence for Mirounga angustirostris produced by RNA sequencing of muscle tissue and cloud-based de novo transcriptome assembly. We annotated 395,102 transcripts, some of which may be novel isoforms, and have identified thousands of genes involved in key physiological processes. This resource provides elephant seal-specific gene sequences, complementing existing metabolite and protein expression studies and enabling future work on molecular pathways regulating adaptations such as fasting, hypoxia, and environmental stress responses in marine mammals.

Metabolic response to a glucagon challenge varies with adiposity and life-history stage in fasting northern elephant seals.

Metabolic adaptations for extended fasting in wildlife prioritize beta-oxidation of lipids and reduced glucose utilization to support energy metabolism. The pancreatic hormone glucagon plays key roles in regulating glycemia and lipid metabolism during fasting in model species but its function in wildlife species adapted for extended fasting is not well understood. Northern elephant seals (NES) undergo natural fasts of 1-3months while under constraints of high nutrient demands including lactation and development. We performed a glucagon challenge on lactating, molting and developing NES, early and late in their natural fasts, to examine the impact of this important regulatory hormone on metabolism. Glucagon caused increases in plasma glucose, insulin, fatty acids, ketones and urea, but the magnitude of these effects varied widely with adiposity and life-history stage. The strong impact of adiposity on glucose and insulin responses suggest a potential role for adipose derived factors in regulating hepatic metabolism and pancreatic sensitivity. Elevations in plasma glucose in response to glucagon were strongly associated with increases in protein catabolism, suggesting negative impacts of elevated glucagon on protein sparing. Glucagon promoted rapid ketone accumulation suggesting that low ketoacid levels in NES reflect low rates of production. These results demonstrate strong metabolic impacts of glucagon and support the idea that glucagon levels are downregulated in the context of metabolic adaptation to extended fasting. These results suggest that the regulation of carbohydrate and lipid metabolism in NES changes with adiposity, fasting duration and under various constraints of nutrient demands.

Gluconeogenesis is associated with high rates of tricarboxylic acid and pyruvate cycling in fasting northern elephant seals.

Animals that endure prolonged periods of food deprivation preserve vital organ function by sparing protein from catabolism. Much of this protein sparing is achieved by reducing metabolic rate and suppressing gluconeogenesis while fasting. Northern elephant seals (Mirounga angustirostris) endure prolonged fasts of up to 3 mo at multiple life stages. During these fasts, elephant seals maintain high levels of activity and energy expenditure associated with breeding, reproduction, lactation, and development while maintaining rates of glucose production typical of a postabsorptive mammal. Therefore, we investigated how fasting elephant seals meet the requirements of glucose-dependent tissues while suppressing protein catabolism by measuring the contribution of glycogenolysis, glycerol, and phosphoenolpyruvate (PEP) to endogenous glucose production (EGP) during their natural 2-mo postweaning fast. Additionally, pathway flux rates associated with the tricarboxylic acid (TCA) cycle were measured specifically, flux through phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate cycling. The rate of glucose production decreased during the fast (F(1,13) = 5.7, P = 0.04) but remained similar to that of postabsorptive mammals. The fractional contributions of glycogen, glycerol, and PEP did not change with fasting; PEP was the primary gluconeogenic precursor and accounted for ∼95% of EGP. This large contribution of PEP to glucose production occurred without substantial protein loss. Fluxes through the TCA cycle, PEPCK, and pyruvate cycling were higher than reported in other species and were the most energetically costly component of hepatic carbohydrate metabolism. The active pyruvate recycling fluxes detected in elephant seals may serve to rectify gluconeogeneic PEP production during restricted anaplerotic inflow in these fasting-adapted animals.

Glucose oxidation and nonoxidative glucose disposal during prolonged fasts of the northern elephant seal pup (Mirounga angustirostris).

Elephant seal weanlings demonstrate rates of endogenous glucose production (EGP) during protracted fasts that are higher than predicted on the basis of mass and time fasting. To determine the nonoxidative and oxidative fate of endogenously synthesized glucose, substrate oxidation, metabolic rate, glycolysis, and EGP were measured in fasting weanlings. Eight weanlings were sampled at 14 days of fasting, and a separate group of nine weanlings was sampled at 49 days of fasting. Metabolic rate was determined via flow-through respirometry, and substrate-specific oxidation was determined from the respiratory quotient and urinary nitrogen measurements. The rate of glucose disposal (Glu((R)(d))) was determined through a primed, constant infusion of [3-(3)H]glucose, and glycolysis was determined from the rate of appearance of (3)H in the body water pool. Glu((R)(d)) was 1.41 ± 0.27 and 0.95 ± 0.21 mmol/min in the early and late fasting groups, respectively. Nearly all EGP went through glycolysis, but the percentage of Glu((R)(d)) oxidized to meet the daily metabolic demand was only 24.1 ± 4.4% and 16.7 ± 5.9% between the early and late fasting groups. Glucose oxidation was consistently less than 10% of the metabolic rate in both groups. This suggests that high rates of EGP do not support substrate provisions for glucose-demanding tissues. It is hypothesized that rates of EGP may be ancillary to the upregulation of the tricarboxylic acid cycle to meet high rates of lipid oxidation while mitigating ketosis.

Sex differences in fuel use and metabolism during development in fasting juvenile northern elephant seals.

Many polygynous, capital breeders exhibit sexual dimorphism with respect to body size and composition. Sexual dimorphism is often facilitated by sex differences in foraging behavior, growth rates and patterns of nutrient deposition during development. In species that undergo extended fasts during development, metabolic strategies for fuel use have the potential to influence future reproductive success by directly impacting somatic growth and acquisition of traits required for successful breeding. We investigated sexual dimorphism associated with metabolic strategies for fasting in developing northern elephant seals. Thirty-one juvenile seals of both sexes were sampled over extended fasts during annual autumn haul-outs. Field metabolic rate (FMR) and the contribution of protein catabolism to energy expenditure were estimated from changes in mass and body composition over 23±5 days of fasting (mean ± s.d.). Protein catabolism was assessed directly in a subset of animals based on urea flux at the beginning and end of the fast. Regulatory hormones and blood metabolites measured included growth hormone, cortisol, thyroxine, triiodothyronine, insulin, glucagon, testosterone, estradiol, glucose, urea and ß-hydroxybutyrate. Males exhibited higher rates of energy expenditure during the fast but spared body protein stores more effectively than females. Rates of protein catabolism and energy expenditure were significantly impacted by hormone levels, which varied between the sexes. These data suggest that sex differences in fuel metabolism and energy expenditure during fasting arise early in juvenile development and may play an important role in the development of adult traits associated with reproductive success.

Decreased expression of adipose CD36 and FATP1 are associated with increased plasma non-esterified fatty acids during prolonged fasting in northern elephant seal pups (Mirounga angustirostris).

The northern elephant seal pup (Mirounga angustirostris) undergoes a 2-3 month post-weaning fast, during which it depends primarily on the oxidation of fatty acids to meet its energetic demands. The concentration of non-esterified fatty acids (NEFAs) increases and is associated with the development of insulin resistance in late-fasted pups. Furthermore, plasma NEFA concentrations respond differentially to an intravenous glucose tolerance test (ivGTT) depending on fasting duration, suggesting that the effects of glucose on lipid metabolism are altered. However, elucidation of the lipolytic mechanisms including lipase activity during prolonged fasting in mammals is scarce. To assess the impact of fasting and glucose on the regulation of lipid metabolism, adipose tissue and plasma samples were collected before and after ivGTTs performed on early (2 weeks, N=5) and late (6-8 weeks; N=8) fasted pups. Glucose administration increased plasma triglycerides and NEFA concentrations in late-fasted seals, but not plasma glycerol. Fasting decreased basal adipose lipase activity by 50%. Fasting also increased plasma lipase activity twofold and decreased the expressions of CD36, FAS, FATP1 and PEPCK-C by 22-43% in adipose tissue. Plasma acylcarnitine profiling indicated that late-fasted seals display higher incomplete LCFA ß-oxidation. Results suggest that long-term fasting induces shifts in the regulation of lipolysis and lipid metabolism associated with the onset of insulin resistance in northern elephant seal pups. Delineation of the mechanisms responsible for this shift in regulation during fasting can contribute to a more thorough understanding of the changes in lipid metabolism associated with dyslipidemia and insulin resistance in mammals.

Water-soluble vitamin homeostasis in fasting northern elephant seals (Mirounga angustirostris) measured by metabolomics analysis and standard methods.

Despite the importance of water-soluble vitamins to metabolism, there is limited knowledge of their serum availability in fasting wildlife. We evaluated changes in water-soluble vitamins in northern elephant seals, a species with an exceptional ability to withstand nutrient deprivation. We used a metabolomics approach to measure vitamins and associated metabolites under extended natural fasts for up to 7 weeks in free-ranging lactating or developing seals. Water-soluble vitamins were not detected with this metabolomics platform, but could be measured with standard assays. Concentrations of measured vitamins varied independently, but all were maintained at detectable levels over extended fasts, suggesting that defense of vitamin levels is a component of fasting adaptation in the seals. Metabolomics was not ideal for generating complete vitamin profiles in this species, but gave novel insights into vitamin metabolism by detecting key related metabolites. For example, niacin level reductions in lactating females were associated with significant reductions in precursors suggesting downregulation of the niacin synthetic pathway. The ability to detect individual vitamins using metabolomics may be impacted by the large number of novel compounds detected. Modifications to the analysis platforms and compound detection algorithms used in this study may be required for improving water-soluble vitamin detection in this and other novel wildlife systems.

A blubber gene expression index for evaluating stress in marine mammals.

Evaluating the impacts of anthropogenic disturbance on free-ranging marine mammal populations, many of which are in decline, requires robust diagnostic markers of physiological stress and health. However, circulating levels of canonical 'stress hormones' such as glucocorticoids, which are commonly used to evaluate animal health, do not capture the complexity of species-specific responses and cannot be easily measured in large, fully aquatic marine mammals. Alternatively, expression of stress-responsive genes in hormone target tissues such as blubber, the specialized subcutaneous adipose tissue that can be manually or remotely sampled from many marine mammals, may be a more informative and sensitive indicator of recent (within 24 h) exposure to stressors. We previously identified genes that were upregulated in the inner blubber of juvenile northern elephant seals during experimental stimulation of the hypothalamic-pituitary-adrenal axis. In this study, we measured baseline expression levels of a subset of these genes in inner blubber of unmanipulated juvenile elephant seals of varying physiological states and correlated them with other stress markers (body condition index, corticosteroid and thyroid hormone levels). Expression of 10 genes, including those associated with lipid metabolism (ACSL1, HMGCS2, CDO1), redox homeostasis (GPX3), adipokine signaling (ADIPOQ), lipid droplet formation (PLIN1, CIDEA) and adipogenesis (DKK1, AZGP1, TGFBI), was described by three principal components and was associated with cortisol and thyroid hormone levels. Significantly, baseline gene expression levels were predictive of circulating hormone levels, suggesting that these markers may be potential indicators of exposure to stressors in marine mammal species that are inaccessible for blood sampling. A similar approach may be used to identify species-specific stress markers in other tissues that can be sampled by remote biopsy dart from free-ranging marine mammals, such as outer blubber and skin.

Extreme hypoxemic tolerance and blood oxygen depletion in diving elephant seals.

Species that maintain aerobic metabolism when the oxygen (O(2)) supply is limited represent ideal models to examine the mechanisms underlying tolerance to hypoxia. The repetitive, long dives of northern elephant seals (Mirounga angustirostris) have remained a physiological enigma as O(2) stores appear inadequate to maintain aerobic metabolism. We evaluated hypoxemic tolerance and blood O(2) depletion by 1) measuring arterial and venous O(2) partial pressure (Po(2)) during dives with a Po(2)/temperature recorder on elephant seals, 2) characterizing the O(2)-hemoglobin (O(2)-Hb) dissociation curve of this species, 3) applying the dissociation curve to Po(2) profiles to obtain %Hb saturation (So(2)), and 4) calculating blood O(2) store depletion during diving. Optimization of O(2) stores was achieved by high venous O(2) loading and almost complete depletion of blood O(2) stores during dives, with net O(2) content depletion values up to 91% (arterial) and 100% (venous). In routine dives (>10 min) Pv(O(2)) and Pa(O(2)) values reached 2-10 and 12-23 mmHg, respectively. This corresponds to So(2) of 1-26% and O(2) contents of 0.3 (venous) and 2.7 ml O(2)/dl blood (arterial), demonstrating remarkable hypoxemic tolerance as Pa(O(2)) is nearly equivalent to the arterial hypoxemic threshold of seals. The contribution of the blood O(2) store alone to metabolic rate was nearly equivalent to resting metabolic rate, and mean temperature remained near 37 degrees C. These data suggest that elephant seals routinely tolerate extreme hypoxemia during dives to completely utilize the blood O(2) store and maximize aerobic dive duration.

Blubber transcriptome responses to repeated ACTH administration in a marine mammal.

Chronic physiological stress impacts animal fitness by catabolizing metabolic stores and suppressing reproduction. This can be especially deleterious for capital breeding carnivores such as marine mammals, with potential for ecosystem-wide effects. However, the impacts and indicators of chronic stress in animals are currently poorly understood. To identify downstream mediators of repeated stress responses in marine mammals, we administered adrenocorticotropic hormone (ACTH) once daily for four days to free-ranging juvenile northern elephant seals (Mirounga angustirostris) to stimulate endogenous corticosteroid release, and compared blubber tissue transcriptome responses to the first and fourth ACTH administrations. Gene expression profiles were distinct between blubber responses to single and repeated ACTH administration, despite similarities in circulating cortisol profiles. We identified 61 and 12 genes that were differentially expressed (DEGs) in response to the first ACTH and fourth administrations, respectively, 24 DEGs between the first and fourth pre-ACTH samples, and 12 DEGs between ACTH response samples from the first and fourth days. Annotated DEGs were associated with functions in redox and lipid homeostasis, suggesting potential negative impacts of repeated stress on capital breeding, diving mammals. DEGs identified in this study are potential markers of repeated stress in marine mammals, which may not be detectable by endocrine profiles alone.

Repeated adrenocorticotropic hormone administration alters adrenal and thyroid hormones in free-ranging elephant seals.

Understanding the physiological response of marine mammals to anthropogenic stressors can inform marine ecosystem conservation strategies. Stress stimulates the activation of the hypothalamic-pituitary-adrenal (HPA) axis and synthesis of glucocorticoid (GC) hormones, which increase energy substrate availability while suppressing energy-intensive processes. Exposure to repeated stressors can potentially affect an animal's ability to respond to and recover from subsequent challenges. To mimic repeated activation of the HPA axis by environmental stressors (or challenges), we administered adrenocorticotropic hormone (ACTH) to free-ranging juvenile northern elephant seals (Mirounga angustirostris; n = 7) once daily for 4 days. ACTH administration induced significant elevation in circulating cortisol and aldosterone levels. The cortisol responses did not vary in magnitude between the first ACTH administration on Day 1 and the last administration on Day 4. In contrast, aldosterone levels remained elevated above baseline for at least 24 h after each ACTH injection, and responses were greater on Day 4 than Day 1. Total triiodothyronine (tT3) levels were decreased on Day 4 relative to Day 1, while reverse triiodothyronine (rT3) concentrations increased relative to baseline on Days 1 and 4 in response to ACTH, indicating a suppression of thyroid hormone production. There was no effect of ACTH on the sex steroid dehydroepiandrosterone. These data suggest that elephant seals are able to mount adrenal responses to multiple ACTH administrations. However, repeated ACTH administration resulted in facilitation of aldosterone secretion and suppression of tT3, which may impact osmoregulation and metabolism, respectively. We propose that aldosterone and tT3 are informative additional indicators of repeated stress in marine mammals.

A sample preparation workflow for adipose tissue shotgun proteomics and proteogenomics.

Animals with large adipose stores, such as marine mammals, may provide insights into the evolution and function of this multifunctional tissue in health and disease. In the absence of sequenced genomes, molecular information can be rapidly obtained by proteomics and transcriptomics, but their application to adipose tissue is hindered by low nucleic acid and protein yields. We sequenced and compared proteomes isolated from the blubber of four elephant seals using phenol and guanidine thiocyanate (Qiazol) or detergent (sodium deoxycholate) buffer. Qiazol recovered more subcellular proteins such as metabolic enzymes, in addition to extracting RNA, facilitating proteogenomic analyses of small lipid-rich tissue biopsies. We also compared proteomics data analysis platforms and found that de novo peptide sequencing improved protein identification sensitivity compared to database search alone. We report sample preparation and data analysis workflows for proteogenomics and a proteome of elephant seal blubber containing 2678 proteins, including many of interest for further functional studies.This article has an associated First Person interview with the first author of the paper.

Adult male northern elephant seals maintain high rates of glucose production during extended breeding fasts.

Many species undergo natural fasts as part of their life histories. Extended fasting is associated with increased ß-oxidation of fatty acids and reduced oxidation of glucose to minimize commitment of body protein to gluconeogenesis. However, the metabolic strategies used to sustain extended fasts simultaneous with high rates of energy expenditure are not well understood. Studies in fasting adult female and weanling northern elephant seals (NES) have revealed high rates of endogenous glucose production (EGP) under constraints of high nutrient demand for lactation or development but relatively low rates of metabolism. These studies revealed low rates of glucose oxidation and high rates of glucose recycling through the Cori cycle. We measured rates of glucose flux in fasting adult male NES to assess how significantly longer fasting durations, higher metabolic rates, and greater rates of muscular activity affect glucose kinetics. We measured glucose turnover in 18 adult males using the clearance of [6-H3] glucose during breeding and molting. Adult male NES maintain high rates of EGP across extended fasts. EGP greatly exceeded estimated needs for glucose-dependent tissues, varied directly with plasma insulin and lactate concentrations, and was inversely related to plasma ketoacid concentrations. Together, these findings suggest that high rates of glucose production and recycling during breeding maintain high blood glucose levels to support glucose-dependent tissues while minimizing production of ketoacids and commitment of protein stores to glucose production.

Effects of oral megestrol acetate administration on the hypothalamic-pituitary-adrenal axis of male bottlenose dolphins (Tursiops truncatus).

OBJECTIVE To evaluate the impact of oral megestrol acetate (MA) administration on adrenal function in male bottlenose dolphins (Tursiops truncatus). DESIGN Serial cross-sectional study. ANIMALS 8 adult male dolphins, all of which were receiving MA at various daily doses (range, 0 to 60 mg, PO) for the control of reproductive behavior. PROCEDURES Blood samples were collected every 2 weeks for 1 year from dolphins trained to voluntarily provide them. Cortisol, ACTH, and other hormone concentrations were measured in serum or plasma via radioimmunoassay or ELISA. Fecal samples, also provided by dolphins voluntarily, were assayed for glucocorticoid metabolite concentrations. Effects of daily MA dose on hormone concentrations were evaluated. RESULTS Daily MA doses as low as 10 mg strongly suppressed cortisol secretion in nearly all dolphins, and except for a single measurement, no dolphin had measurable serum concentrations at doses ≥ 20 mg. Variations in serum cortisol concentration were unrelated to season but were directly related to ACTH concentrations, suggesting primary effects upstream of the adrenal gland. Cessation of MA administration resulted in almost immediate restoration of measurable serum cortisol concentrations, although concentrations continued to rise in a few dolphins over the following weeks to months. CONCLUSIONS AND CLINICAL RELEVANCE Caution should be exercised when administering MA to control reproductive behavior in male dolphins. Because the hypothalamic-pituitary-adrenal axis appeared to be sensitive to even small doses of MA in dolphins, duration of treatment may be the most critical consideration.

The environment as a driver of immune and endocrine responses in dolphins (Tursiops truncatus).

Immune and endocrine responses play a critical role in allowing animals to adjust to environmental perturbations. We measured immune and endocrine related markers in multiple samples from individuals from two managed-care care dolphin groups (n = 82 samples from 17 dolphins and single samples collected from two wild dolphin populations: Indian River Lagoon, (IRL) FL (n = 26); and Charleston, (CHS) SC (n = 19). The immune systems of wild dolphins were more upregulated than those of managed-care-dolphins as shown by higher concentrations of IgG and increases in lysozyme, NK cell function, pathogen antibody titers and leukocyte cytokine transcript levels. Collectively, managed-care care dolphins had significantly lower levels of transcripts encoding pro-inflammatory cytokine TNF, anti-viral MX1 and INFα and regulatory IL-10. IL-2Rα and CD69, markers of lymphocyte activation, were both lower in managed-care care dolphins. IL-4, a cytokine associated with TH2 activity, was lower in managed-care care dolphins compared to the free-ranging dolphins. Differences in immune parameters appear to reflect the environmental conditions under which these four dolphin populations live which vary widely in temperature, nutrition, veterinary care, pathogen/contaminant exposures, etc. Many of the differences found were consistent with reduced pathogenic antigenic stimulation in managed-care care dolphins compared to wild dolphins. Managed-care care dolphins had relatively low TH2 lymphocyte activity and fewer circulating eosinophils compared to wild dolphins. Both of these immunologic parameters are associated with exposure to helminth parasites which is uncommon in managed-care care dolphins. Less consistent trends were observed in a suite of hormones but significant differences were found for cortisol, ACTH, total T4, free T3, and epinephrine. While the underlying mechanisms are likely multiple and complex, the marked differences observed in the immune and endocrine systems of wild and managed-care care dolphins appear to be shaped by their environment.