Michael S. Tift

Apnea stimulates the adaptive response to oxidative stress in elephant seal pups.

SUMMARY Extended breath-hold (apnea) bouts are routine during diving and sleeping in seals. These apneas result in oxygen store depletion and blood flow redistribution towards obligatory oxygen-dependent tissues, exposing seals to critical levels of ischemia and hypoxemia. The subsequent reperfusion/reoxygenation has the potential to increase oxidant production and thus oxidative stress. The contributions of extended apnea to oxidative stress in adapted mammals are not well defined. To address the hypothesis that apnea in seals is not associated with increased oxidative damage, blood samples were collected from northern elephant seal pups (N=6) during eupnea, rest- and voluntary submersion-associated apneas, and post-apnea (recovery). Plasma 4-hydroxynonenal (HNE), 8-isoprostanes (8-isoPGF2α), nitrotyrosine (NT), protein carbonyls, xanthine and hypoxanthine (HX) levels, along with xanthine oxidase (XO) activity, were measured. Protein content of XO, superoxide dismutase 1 (Cu,ZnSOD), catalase and myoglobin (Mb), as well as the nuclear content of hypoxia inducible factor 1α (HIF-1α) and NF-E2-related factor 2 (Nrf2), were measured in muscle biopsies collected before and after the breath-hold trials. HNE, 8-iso PGF2α, NT and protein carbonyl levels did not change among eupnea, apnea or recovery. XO activity and HX and xanthine concentrations were increased at the end of the apneas and during recovery. Muscle protein content of XO, CuZnSOD, catalase, Mb, HIF-1α and Nrf2 increased 25–70% after apnea. Results suggest that rather than inducing the damaging effects of hypoxemia and ischemia/reperfusion that have been reported in non-diving mammals, apnea in seals stimulates the oxidative stress and hypoxic hormetic responses, allowing these mammals to cope with the potentially detrimental effects associated with this condition.

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

SUMMARY 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 β-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.

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.

Insulin and GLP-1 infusions demonstrate the onset of adipose-specific insulin resistance in a large fasting mammal: potential glucogenic role for GLP-1

Prolonged food deprivation increases lipid oxidation and utilization, which may contribute to the onset of the insulin resistance associated with fasting. Because insulin resistance promotes the preservation of glucose and oxidation of fat, it has been suggested to be an adaptive response to food deprivation. However, fasting mammals exhibit hypoinsulinemia, suggesting that the insulin resistance‐like conditions they experience may actually result from reduced pancreatic sensitivity to glucose/capacity to secrete insulin. To determine whether fasting results in insulin resistance or in pancreatic dysfunction, we infused early‐ and late‐fasted seals (naturally adapted to prolonged fasting) with insulin (0.065 U/kg), and a separate group of late‐fasted seals with low (10 pmol/L per kg) or high (100 pmol/L per kg) dosages of glucagon‐like peptide‐1 (GLP‐1) immediately following a glucose bolus (0.5 g/kg), and measured the systemic and cellular responses. Because GLP‐1 facilitates glucose‐stimulated insulin secretion, these infusions provide a method to assess pancreatic insulin‐secreting capacity. Insulin infusions increased the phosphorylation of insulin receptor and Akt in adipose and muscle of early‐ and late‐fasted seals; however, the timing of the signaling response was blunted in adipose of late‐fasted seals. Despite the dose‐dependent increases in insulin and increased glucose clearance (high dose), both GLP‐1 dosages produced increases in plasma cortisol and glucagon, which may have contributed to the glucogenic role of GLP‐1. Results suggest that fasting induces adipose‐specific insulin resistance in elephant seal pups, while maintaining skeletal muscle insulin sensitivity, and therefore suggests that the onset of insulin resistance in fasting mammals is an evolved response to cope with prolonged food deprivation.

Development enhances hypometabolism in northern elephant seal pups (Mirounga angustirostris)

Investigation into the development of oxygen storage capacity in air-breathing marine predators has been performed, but little is known about the development of regulatory factors that influence oxygen utilization. Strategies for efficiently using oxygen stores should enable marine predators to optimize time spent foraging underwater.We describe the developmental patterns of oxygen use during voluntary breath-holds in northern elephant seals (Mirounga angustirostris) at 2 and 7 weeks post-weaning. We measured 1) changes in oxygen consumption (VO2), and 2) changes in venous pH, partial pressure of oxygen (pO2), haemoglobin saturation (sO2), oxygen content (O2ct), partial pressure of carbon dioxide (pCO2), haematocrit (Hct) and total haemoglobin (tHb). To examine the effect of the dive response on the development of oxygen utilization, voluntary breath-hold experiments were conducted in and out of water.Suppression of VO2 during voluntary breath-holds increased significantly between 2 and 7 weeks post-weaning, reaching a maximum suppression of 53% below resting metabolic rate and 56% below Kleibers standard metabolic rate. From 2 to 7 weeks post-weaning, breath-hold VO2 was reduced by 52%. Between the two age classes, this equates to a mean breath-hold VO2 reduction of 16% from resting VO2. Breath-hold VO2 also declined with increasing breath-hold duration, but there was no direct effect of voluntary submergence on reducing VO2.Age did not influence rates of venous pO2 depletion during breath-holds. However, voluntary submergence did result in slower pO2 depletion rates when compared to voluntary terrestrial apnoeas. The differences in whole body VO2 during breath-holds (measured at recovery) and venous pO2 (reflective of tissue O2-use measured during breath-holds), likely reflects metabolic suppression in hypoxic, vasoconstricted tissues.Consistent pCO2 values at the end of all voluntary breath-holds (59.0 ± 0.7 mmHg) suggests the physiological cue for stimulating respiration in northern elephant seal pups is the accumulation of CO2.Oxygen storage capacity and metabolic suppression directly limit diving capabilities and may influence foraging success in low-weaning weight seals forced to depart to sea prior to achieving full developmental diving capacity.

High-density lipoprotein remains elevated despite reductions in total cholesterol in fasting adult male elephant seals (Mirounga angustirostris).

We examined changes in lipid profiles of 40 adult northern elephant seal bulls over the 3-month breeding fast and the 1-month molting fast to investigate impacts of fasting on serum total cholesterol (TC), triglycerides (TG) and lipoproteins. Total cholesterol and low-density lipoprotein (LDL) levels were initially high (3930 ± 190mgL(-1)and 1610 ± 170mgL(-1), respectively) and decreased significantly over the breeding season. Total cholesterol and LDL declined significantly with adipose tissue reserves (p<0.001), and LDL levels as low as 43 mgL(-1) were measured in seals late in the breeding fast. Less dramatic but similar changes in lipid metabolism were observed across the molting fast. High-density lipoproteins (HDL) remained consistently elevated (>1750 mgL(-1)) suggesting that elephant seals defend HDL concentrations, despite significant depletion of TC and LDL across the breeding fast. Triglyceride levels were significantly higher during the molt, consistent with lower rates of lipid oxidation needed to meet metabolic energy demands during this period. The maintenance of HDL during breeding is consistent with its role in delivering cholesterol from adipose tissue for steroidogenesis and spermatogenesis and potentially mitigates oxidative stress associated with fasting.

Adrenal sensitivity to stress is maintained despite variation in baseline glucocorticoids in moulting seals

Select hormones, like glucocorticoids, could be informative markers of stress in animals. To be useful, however, baseline and stressed state hormone concentrations must be described. We therefore evaluated the timing and magnitude of stress hormone release by simulating an acute stressor in a marine mammal, the northern elephant seal.

Elevated carboxyhemoglobin in a marine mammal, the northern elephant seal

Low concentrations of endogenous carbon monoxide (CO), generated primarily through degradation of heme from heme-proteins, have been shown to maintain physiological function of organs and to exert cytoprotective effects. However, high concentrations of carboxyhemoglobin (COHb), formed by CO binding to hemoglobin, potentially prevent adequate O2 delivery to tissues by lowering arterial O2 content. Elevated heme-protein concentrations, as found in marine mammals, are likely associated with greater heme degradation, more endogenous CO production and, consequently, elevated COHb concentrations. Therefore, we measured COHb in elephant seals, a species with large blood volumes and elevated hemoglobin and myoglobin concentrations. The levels of COHb were positively related to the total hemoglobin concentration. The maximum COHb value was 10.4% of total hemoglobin concentration. The mean (±s.e.m.) value in adult seals was 8.7±0.3% (N=6), while juveniles and pups (with lower heme-protein contents) had lower mean COHb values of 7.6±0.2% and 7.1±0.3%, respectively (N=9 and N=9, respectively). Serial samples over several hours revealed little to no fluctuation in COHb values. This consistent elevation in COHb suggests that the magnitude and/or rate of heme-protein turnover is much higher than in terrestrial mammals. The maximum COHb values from this study decrease total body O2 stores by 7%, thereby reducing the calculated aerobic dive limit for this species. However, the constant presence of elevated CO in blood may also protect against potential ischemia–reperfusion injury associated with the extreme breath-holds of elephant seals. We suggest the elephant seal represents an ideal model for understanding the potential cytoprotective effects, mechanisms of action and evolutionary adaptation associated with chronically elevated concentrations of endogenously produced CO.

Regional variability in diving physiology and behavior in a widely distributed air-breathing marine predator, the South American sea lion (Otaria byronia).

ABSTRACT Our understanding of how air-breathing marine predators cope with environmental variability is limited by our inadequate knowledge of their ecological and physiological parameters. Because of their wide distribution along both coasts of the sub-continent, South American sea lions (Otaria byronia) provide a valuable opportunity to study the behavioral and physiological plasticity of a marine predator in different environments. We measured the oxygen stores and diving behavior of South American sea lions throughout most of its range, allowing us to demonstrate that diving ability and behavior vary across its range. We found no significant differences in mass-specific blood volumes of sea lions among field sites and a negative relationship between mass-specific oxygen storage and size, which suggests that exposure to different habitats and geographical locations better explains oxygen storage capacities and diving capability in South American sea lions than body size alone. The largest animals in our study (individuals from Uruguay) were the shallowest and shortest duration divers, and had the lowest mass-specific total body oxygen stores, while the deepest and longest duration divers (individuals from southern Chile) had significantly larger mass-specific oxygen stores, despite being much smaller animals. Our study suggests that the physiology of air-breathing diving predators is not fixed, but that it can be adjusted, to a certain extent, depending on the ecological setting and or habitat. These adjustments can be thought of as a ‘training effect’: as the animal continues to push its physiological capacity through greater hypoxic exposure, its breath-holding capacity increases. Summary: Regional variation in diving ability and behavior of the South American sea lion suggests that exposure to different habitats and geographical locations better explains oxygen storage capacities and diving capability than body size alone.

Flipper stroke rate and venous oxygen levels in free-ranging California sea lions

ABSTRACT The depletion rate of the blood oxygen store, development of hypoxemia and dive capacity are dependent on the distribution and rate of blood oxygen delivery to tissues while diving. Although blood oxygen extraction by working muscle would increase the blood oxygen depletion rate in a swimming animal, there is little information on the relationship between muscle workload and blood oxygen depletion during dives. Therefore, we examined flipper stroke rate, a proxy of muscle workload, and posterior vena cava oxygen profiles in four adult female California sea lions (Zalophus californianus) during foraging trips at sea. Flipper stroke rate analysis revealed that sea lions minimized muscle metabolism with a stroke–glide strategy when diving, and exhibited prolonged glides during the descent of deeper dives (>100 m). During the descent phase of these deep dives, 55±21% of descent was spent gliding, with the longest glides lasting over 160 s and covering a vertical distance of 340 m. Animals also consistently glided to the surface from 15 to 25 m depth during these deeper dives. Venous hemoglobin saturation (SO2) profiles were highly variable throughout dives, with values occasionally increasing during shallow dives. The relationship between SO2 and flipper stroke rate was weak during deeper dives, while this relationship was stronger during shallow dives. We conclude that (1) the depletion of oxygen in the posterior vena cava in deep-diving sea lions is not dependent on stroke effort, and (2) stroke–glide patterns during dives contribute to a reduction of muscle metabolic rate. Summary: Flipper stroke rate has little impact on posterior vena caval oxygen depletion patterns in diving California sea lions. Use of a stroke–glide strategy also contributes to minimization of work effort.