Relationship between red blood cell lifespan and endogenous carbon monoxide in the common bottlenose dolphin and beluga.

Certain deep-diving marine mammals [i.e., northern elephant seal (Mirounga angustirostris), Weddell seal (Leptonychotes weddellii)] have blood carbon monoxide (CO) levels that are comparable with those of chronic cigarette smokers. Most CO produced in humans is a byproduct of heme degradation, which is released when red blood cells (RBCs) are destroyed. Elevated CO can occur in humans when RBC lifespan decreases. The contribution of RBC turnover to CO concentrations in marine mammals is unknown. Here, we report the first RBC lifespans in two healthy marine mammal species with different diving capacities and heme stores, the shallow-diving bottlenose dolphin (Tursiops truncatus) and deep-diving beluga whale (Delphinapterus leucas), and we relate the lifespans to the levels of CO in blood and breath. The belugas, with high blood heme stores, had the longest mean RBC lifespan compared with humans and bottlenose dolphins. Both cetacean species were found to have three times higher blood CO content compared with humans. The estimated CO production rate from heme degradation indicates some marine mammals may have additional mechanisms for CO production, or delay CO removal from the body, potentially from long-duration breath-holds.NEW & NOTEWORTHY This is the first study to determine the red blood cell lifespan in a marine mammal species. High concentrations of carbon monoxide (CO) were found in the blood of bottlenose dolphins and in the blood and breath of belugas compared with healthy humans. Red blood cell turnover accounted for these high levels in bottlenose dolphins, but there may be alternative mechanisms of endogenous CO production that are contributing to the CO concentrations observed in belugas.

Oral Carbon Monoxide Enhances Autophagy Modulation in Prostate, Pancreatic, and Lung Cancers.

Modulation of autophagy, specifically its inhibition, stands to transform the capacity to effectively treat a broad range of cancers. However, the clinical efficacy of autophagy inhibitors has been inconsistent. To delineate clinical and epidemiological features associated with autophagy inhibition and a positive oncological clinical response, a retrospective analysis of patients is conducted treated with hydroxychloroquine, a known autophagy inhibitor. A direct correlation between smoking status and inhibition of autophagy with hydroxychloroquine is identified. Recognizing that smoking is associated with elevated circulating levels of carbon monoxide (CO), it is hypothesized that supplemental CO can amplify autophagy inhibition. A novel, gas-entrapping material containing CO in a pre-clinical model is applied and demonstrated that CO can dramatically increase the cytotoxicity of autophagy inhibitors and significantly inhibit the growth of tumors when used in combination. These data support the notion that safe, therapeutic levels of CO can markedly enhance the efficacy of autophagy inhibitors, opening a promising new frontier in the quest to improve cancer therapies.

Preserved peak exercise capacity in Andean highlanders with excessive erythrocytosis both before and after isovolumic hemodilution.

In chronic mountain sickness (CMS), increased blood oxygen (O2)-carrying capacity due to excessive erythrocytosis (EE, [Hb] ≥ 21 g/dL) could be offset, especially during exercise by both impaired cardiac output (Q̇t) and O2 diffusion limitation in lungs and muscle. We hypothesized that EE results in reduced peak V̇o2 despite increased blood O2-carrying capacity, and that isovolumic hemodilution (IVHD) improves exercise capacity. In 14 male residents of Cerro de Pasco, Peru (4,340 m), six with and eight without EE, we measured peak cycle-exercise capacity, V̇o2, Q̇t, arterial blood gas parameters, and (resting) blood volume. This was repeated for participants with EE after IVHD, reducing hematocrit by 20% (from 67% to 53%). From these data, we quantified the major O2 transport pathway components (ventilation, pulmonary alveolar-capillary diffusion, Q̇t, and blood-muscle mitochondria diffusion). Participants with EE had similar peak V̇o2, systemic O2 delivery, and O2 extraction as non-EE controls, however, with lower Q̇t and higher arterial [O2]. After IVHD, peak V̇o2 was preserved (but not enhanced), with lower O2 delivery (despite higher Q̇t) balanced by greater O2 extraction. The considerable variance in exercise capacity across the 14 individuals was explained essentially completely by differences in both pulmonary and muscle O2 diffusional conductances and not by any differences in ventilation, [Hb], nor Q̇t. In conclusion, EE does not result in lower peak V̇o2 in Andean males, and IVHD maintains, but does not enhance, exercise capacity.NEW & NOTEWORTHY Male Andean highlanders with and without excessive erythrocytosis (EE) have similar peak V̇o2 at 4,340 m, with higher arterial [O2] in EE and lower cardiac output (Q̇t), thus maintaining similar O2 delivery. Peak V̇o2 in participants with EE was unaffected by isovolumic hemodilution (hematocrit reduced from 67% to 53%), with lower O2 delivery balanced by slightly increased Q̇t and greater O2 extraction. Differences in lung and muscle diffusing capacity, and not hematocrit variation, accounted for essentially all interindividual variance in peak V̇o2.

Remarkable consistency of spinal cord microvasculature in highly adapted diving odontocetes.

Odontocetes are breath-hold divers with a suite of physiological, anatomical, and behavioral adaptations that are highly derived and vastly different from those of their terrestrial counterparts. Because of these adaptations for diving, odontocetes were originally thought to be exempt from the harms of nitrogen gas embolism while diving. However, recent studies have shown that these mammals may alter their dive behavior in response to anthropogenic sound, leading to the potential for nitrogen supersaturation and bubble formation which may cause decompression sickness in the central nervous system (CNS). We examined the degree of interface between blood, gases, and neural tissues in the spinal cord by quantifying its microvascular characteristics in five species of odontocetes (Tursiops truncatus, Delphinus delphis, Grampus griseus, Kogia breviceps, and Mesoplodon europaeus) and a model terrestrial species (the pig-Sus scrofa domesticus) for comparison. This approach allowed us to compare microvascular characteristics (microvascular density, branching, and diameter) at several positions (cervical, thoracic, and lumbar) along the spinal cord from odontocetes that are known to be either deep or shallow divers. We found no significant differences (p < 0.05 for all comparisons) in microvessel density (9.30-11.18%), microvessel branching (1.60-2.12 branches/vessel), or microvessel diameter (11.83-16.079 µm) between odontocetes and the pig, or between deep and shallow diving odontocete species. This similarity of spinal cord microvasculature anatomy in several species of odontocetes as compared to the terrestrial mammal is in contrast to the wide array of remarkable physio-anatomical adaptations marine mammals have evolved within their circulatory system to cope with the physiological demands of diving. These results, and other studies on CNS lipids, indicate that the spinal cords of odontocetes do not have specialized features that might serve to protect them from Type II DCS.

Time Domains of Hypoxia Responses and -Omics Insights.

The ability to respond rapidly to changes in oxygen tension is critical for many forms of life. Challenges to oxygen homeostasis, specifically in the contexts of evolutionary biology and biomedicine, provide important insights into mechanisms of hypoxia adaptation and tolerance. Here we synthesize findings across varying time domains of hypoxia in terms of oxygen delivery, ranging from early animal to modern human evolution and examine the potential impacts of environmental and clinical challenges through emerging multi-omics approaches. We discuss how diverse animal species have adapted to hypoxic environments, how humans vary in their responses to hypoxia (i.e., in the context of high-altitude exposure, cardiopulmonary disease, and sleep apnea), and how findings from each of these fields inform the other and lead to promising new directions in basic and clinical hypoxia research.

Ontogeny of Carbon Monoxide-Related Gene Expression in a Deep-Diving Marine Mammal.

Marine mammals such as northern elephant seals (NES) routinely experience hypoxemia and ischemia-reperfusion events to many tissues during deep dives with no apparent adverse effects. Adaptations to diving include increased antioxidants and elevated oxygen storage capacity associated with high hemoprotein content in blood and muscle. The natural turnover of heme by heme oxygenase enzymes (encoded by HMOX1 and HMOX2) produces endogenous carbon monoxide (CO), which is present at high levels in NES blood and has been shown to have cytoprotective effects in laboratory systems exposed to hypoxia. To understand how pathways associated with endogenous CO production and signaling change across ontogeny in diving mammals, we measured muscle CO and baseline expression of 17 CO-related genes in skeletal muscle and whole blood of three age classes of NES. Muscle CO levels approached those of animals exposed to high exogenous CO, increased with age, and were significantly correlated with gene expression levels. Muscle expression of genes associated with CO production and antioxidant defenses (HMOX1, BVR, GPX3, PRDX1) increased with age and was highest in adult females, while that of genes associated with protection from lipid peroxidation (GPX4, PRDX6, PRDX1, SIRT1) was highest in adult males. In contrast, muscle expression of mitochondrial biogenesis regulators (PGC1A, ESRRA, ESRRG) was highest in pups, while genes associated with inflammation (HMOX2, NRF2, IL1B) did not vary with age or sex. Blood expression of genes involved in regulation of inflammation (IL1B, NRF2, BVR, IL10) was highest in pups, while HMOX1, HMOX2 and pro-inflammatory markers (TLR4, CCL4, PRDX1, TNFA) did not vary with age. We propose that ontogenetic upregulation of baseline HMOX1 expression in skeletal muscle of NES may, in part, underlie increases in CO levels and expression of genes encoding antioxidant enzymes. HMOX2, in turn, may play a role in regulating inflammation related to ischemia and reperfusion in muscle and circulating immune cells. Our data suggest putative ontogenetic mechanisms that may enable phocid pups to transition to a deep-diving lifestyle, including high baseline expression of genes associated with mitochondrial biogenesis and immune system activation during postnatal development and increased expression of genes associated with protection from lipid peroxidation in adulthood.

Evolved increases in hemoglobin-oxygen affinity and the Bohr effect coincided with the aquatic specialization of penguins.

Dive capacities of air-breathing vertebrates are dictated by onboard O2 stores, suggesting that physiologic specialization of diving birds such as penguins may have involved adaptive changes in convective O2 transport. It has been hypothesized that increased hemoglobin (Hb)-O2 affinity improves pulmonary O2 extraction and enhances the capacity for breath-hold diving. To investigate evolved changes in Hb function associated with the aquatic specialization of penguins, we integrated comparative measurements of whole-blood and purified native Hb with protein engineering experiments based on site-directed mutagenesis. We reconstructed and resurrected ancestral Hb representing the common ancestor of penguins and the more ancient ancestor shared by penguins and their closest nondiving relatives (order Procellariiformes, which includes albatrosses, shearwaters, petrels, and storm petrels). These two ancestors bracket the phylogenetic interval in which penguin-specific changes in Hb function would have evolved. The experiments revealed that penguins evolved a derived increase in Hb-O2 affinity and a greatly augmented Bohr effect (i.e., reduced Hb-O2 affinity at low pH). Although an increased Hb-O2 affinity reduces the gradient for O2 diffusion from systemic capillaries to metabolizing cells, this can be compensated by a concomitant enhancement of the Bohr effect, thereby promoting O2 unloading in acidified tissues. We suggest that the evolved increase in Hb-O2 affinity in combination with the augmented Bohr effect maximizes both O2 extraction from the lungs and O2 unloading from the blood, allowing penguins to fully utilize their onboard O2 stores and maximize underwater foraging time.

Adaptive Potential of the Heme Oxygenase/Carbon Monoxide Pathway During Hypoxia.

Heme oxygenase (HO) enzymes catalyze heme into biliverdin, releasing carbon monoxide (CO) and iron into circulation. These byproducts of heme degradation can have potent cytoprotective effects in the face of stressors such as hypoxia and ischemia-reperfusion events. The potential for exogenous use of CO as a therapeutic agent has received increasing attention throughout the past few decades. Further, HO and CO are noted as putatively adaptive in diving mammals and certain high-altitude human populations that are frequently exposed to hypoxia and/or ischemia-reperfusion events, suggesting that HO and endogenous CO afford an evolutionary advantage for hypoxia tolerance and are critical in cell survival and injury avoidance. Our goal is to describe the importance of examining HO and CO in several systems, the physiological links, and the genetic factors that underlie variation in the HO/CO pathway. Finally, we emphasize the ways in which evolutionary perspectives may enhance our understanding of the HO/CO pathway in the context of diverse clinical settings.

Stroke effort and relative lung volume influence heart rate in diving sea lions.

The dive response, bradycardia (decreased heart rate) and peripheral vasoconstriction, is the key mechanism allowing breath-hold divers to perform long-duration dives while actively swimming and hunting prey. This response is variable and modulated by factors such as dive duration, depth, exercise and cognitive control. This study assessed the potential role of exercise and relative lung volume in the regulation of heart rate (fH) during dives of adult female California sea lions instrumented with electrocardiogram (ECG), depth and tri-axial acceleration data loggers. A positive relationship between activity (minimum specific acceleration) and fH throughout dives suggested increased muscle perfusion associated with exercise. However, apart from late ascent, fH during dives was still less than or equal to resting fH (on land). In addition, the activity-fH relationship was weaker in long, deep dives consistent with prioritization of blood oxygen conservation over blood oxygen delivery to muscle in those dives. Pulmonary stretch receptor reflexes may also contribute to fH regulation as fH profiles generally paralleled changes in relative lung volume, especially in shallower dives and during early descent and late ascent of deeper dives. Overall, these findings support the concept that both exercise and pulmonary stretch receptor reflexes may influence the dive response in sea lions.

The extra burden of motherhood: reduced dive duration associated with pregnancy status in a deep-diving mammal, the northern elephant seal.

The cost of pregnancy is hard to study in marine mammals, particularly in species that undergo pregnancy while diving continuously at sea such as elephant seals (genus Mirounga). We analysed the diving behaviour of confirmed pregnant and non-pregnant northern elephant seals (M. angustirostris, n = 172) and showed that after an initial continuous increase in dive duration, dives of pregnant females become shorter after week 17. The reasons for this reduction in dive duration remain unknown, but we hypothesize that increased fetal demand for oxygen could be the cause. Our findings reveal an opportunity to explore the use of biologging data to investigate pregnancy status of free-ranging marine mammals and factors that could affect pregnancy success.

Anterior vena caval oxygen profiles in a deep-diving California sea lion: arteriovenous shunts, a central venous oxygen store and oxygenation during lung collapse.

Deep-diving California sea lions (Zalophus californianus) can maintain arterial hemoglobin saturation (SO2 ) above 90% despite lung collapse (lack of gas exchange) and extremely low posterior vena caval SO2  in the middle of the dive. We investigated anterior vena caval PO2 and SO2  during dives of an adult female sea lion to investigate two hypotheses: (1) posterior vena caval SO2  is not representative of the entire venous oxygen store and (2) a well-oxygenated (arterialized) central venous oxygen reservoir might account for maintenance of arterial SO2  during lung collapse. During deep dives, initial anterior vena caval SO2  was elevated at 83.6±8.4% (n=102), presumably owing to arteriovenous shunting. It remained high until the bottom phase of the dive and then decreased during ascent, whereas previously determined posterior vena caval SO2  declined during descent and then often increased during ascent. These divergent patterns confirmed that posterior vena caval SO2  was not representative of the entire venous oxygen store. Prior to and early during descent of deep dives, the high SO2  values of both the anterior and posterior venae cavae may enhance arterialization of a central venous oxygen store. However, anterior vena caval SO2  values at depths beyond lung collapse reached levels as low as 40%, making it unlikely that even a completely arterialized central venous oxygen store could account for maintenance of high arterial SO2 These findings suggest that maintenance of high arterial SO2  during deep dives is due to persistence of some gas exchange at depths beyond presumed lung collapse.

Heart rate regulation in diving sea lions: the vagus nerve rules.

Recent publications have emphasized the potential generation of morbid cardiac arrhythmias secondary to autonomic conflict in diving marine mammals. Such conflict, as typified by cardiovascular responses to cold water immersion in humans, has been proposed to result from exercise-related activation of cardiac sympathetic fibers to increase heart rate, combined with depth-related changes in parasympathetic tone to decrease heart rate. After reviewing the marine mammal literature and evaluating heart rate profiles of diving California sea lions (Zalophus californianus), we present an alternative interpretation of heart rate regulation that de-emphasizes the concept of autonomic conflict and the risk of morbid arrhythmias in marine mammals. We hypothesize that: (1) both the sympathetic cardiac accelerator fibers and the peripheral sympathetic vasomotor fibers are activated during dives even without exercise, and their activities are elevated at the lowest heart rates in a dive when vasoconstriction is maximal, (2) in diving animals, parasympathetic cardiac tone via the vagus nerve dominates over sympathetic cardiac tone during all phases of the dive, thus producing the bradycardia, (3) adjustment in vagal activity, which may be affected by many inputs, including exercise, is the primary regulator of heart rate and heart rate fluctuations during diving, and (4) heart beat fluctuations (benign arrhythmias) are common in marine mammals. Consistent with the literature and with these hypotheses, we believe that the generation of morbid arrhythmias because of exercise or stress during dives is unlikely in marine mammals.

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

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.

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

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.

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.

Causes of death in preweaned northern elephant seal pups (Mirounga angustirostris, Gill, 1866), Año Nuevo State Reserve, California, 2012.

During an ongoing physiological ecology study on pups and adult female northern elephant seals (Mirounga angustirostris, Gill, 1866) on the mainland rookery at Año Nuevo State Reserve (California), an opportunity was afforded to collect fresh dead pups for parasitology and necropsy. The investigation was undertaken to delineate the causes of death of northern elephant seals recovered from Año Nuevo State Reserve. Prior to this study, there was no evidence of increased mortality or health problems on this rookery. Necropsies, histology, and ancillary diagnostic studies were conducted on 21 fresh dead preweaned pups. Ages ranged from 1 stillbirth to pups approximately 2 weeks of age. Gross lesions included varying degrees of bruising, hemorrhage, lacerations, and fractures attributed to blunt force trauma to the head, chest, and/or abdomen in 16 pups; starvation in 6 pups; bite wounds in 2 pups; generalized icterus in 2 pups; presumptive drowning in 2 pups; and 1 stillbirth. Most pups had multiple gross lesions. Following light microscopic examination, pups could be assigned into 4 general diagnostic categories: 1) trauma, 2) nutritional status, 3) infectious conditions, and 4) congenital anomalies. This investigation of preweaned pup mortality of northern elephant seals in California further refines diagnostic categories for perinatal pup mortality.

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 Kleiber's 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.

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 pM/kg) or high (100 pM/kg) dosages of glucagon-like peptide-1 (GLP-1) immediately following a glucose bolus (0.5g/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.