Available online at www.sciencedirect.

com

General and Comparative Endocrinology 155 (2008) 352358 www.elsevier.com/locate/ygcen

Variability in leptin and adrenal response in juvenile Steller sea lions (Eumetopias jubatus) to adrenocorticotropic hormone (ACTH) in dierent seasons
Kendall L. Mashburn *, Shannon Atkinson
University of Alaska, Fairbanks and Alaska SeaLife Center, P.O. Box 1329, 301 Railway Avenue, Seward, AK 99664, USA Received 2 February 2007; revised 10 May 2007; accepted 26 May 2007 Available online 2 June 2007

Abstract Eight free-ranging juvenile Steller sea lions (SSL; 6 males, 2 females; 1420 months) temporarily held under ambient conditions at the Alaska SeaLife Center were physiologically challenged through exogenous administration of adrenocorticotropic hormone (ACTH). Four individuals (3 males, 1 female) underwent ACTH challenge in each of two seasons, summer and winter. Following ACTH injection serial blood and fecal samples were collected for up to 3 and 96 h, respectively. A radioimmunoassay (RIA) was validated for leptin, and using a previously validated RIA for cortisol, collected sera were analyzed for both cortisol and leptin. ACTH injection resulted in a 2.9fold increase (P = 0.164) in leptin which preceded a 3.2-fold increase (P = 0.0290) in cortisol by 105 min in summer. In winter, a 1.7-fold increase in leptin (P = 0.020) preceded a 2.1-fold increase (P = 0.001) in serum cortisol by 45 min. Mean fecal corticosteroid maxima were 10.4 and 16.7-fold above baseline 28 and 12 h post-injection and returned to baseline 52 and 32 h post-injection, in summer and winter, respectively. Data indicate acute activity in juvenile adrenal glands is detectable in feces approximately 1224 h post-stimulus in either season, with a duration of approximately 40 h in summer and 20 h in winter. Changes in serum cortisol proved statistically signicant both seasons and elevated concentrations were detected by 30 min post-stimulus (baseline 64.8 4.2; peak 209.5 18.3 ng/ml: summer; baseline 87.0 15.7; peak 237.6 10.0 ng/ml: winter), whereas the changes that occurred in serum leptin proved to be significant only in winter (baseline 6.4 0.6; peak 18.7 7.0 ng/ml: summer; baseline 4.2 0.5; peak 7.5 0.6 ng/ml: winter). Changes in fecal corticosteroids proved signicant only in summer (baseline 117.8 36.7; peak 1219.3 298.4 ng/g, P = 0.038: summer; baseline 71.8 13.7; peak 1198.6 369.9 ng/g, P = 0.053: winter) due to a high degree of individual variability in winter months. The data indicate that ACTH stimulates leptin production earlier than cortisol in both summer and winter, and that while the leptin response appears most variable in summer, fecal corticosteroids are most variable in winter. 2007 Elsevier Inc. All rights reserved.
Keywords: Leptin; Cortisol; Corticosterone; ACTH; Stress; Eumetopias jubatus; Steller sea lion; Juvenile

1. Introduction Steller sea lions (SSL) range along the Pacic rim from central California, through the Gulf of Alaska, along the Aleutian archipelago and the Russian far east to northern Japan. Genetic data led to the division of the population of SSL into two distinct stocks, Western and Eastern, at a
Corresponding author. Fax: +1 907 224 6320. E-mail address: kendall_mashburn@alaskasealife.org (K.L. Mashburn). 0016-6480/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2007.05.030
*

geographic location corresponding to 144W at Cape Suckling, Alaska (Bickham et al., 1996; Loughlin, 1997). Both stocks were listed as threatened under the US Endangered Species Act (ESA) in 1990. While the Eastern stock of SSL has increased slowly but steadily (Calkins et al., 1999; Sease et al., 2001), the Western stock has declined by over 80% (Loughlin et al., 1992; Sease et al., 2001). In 1997, the Western stock of SSL was listed as endangered and the Eastern stock remained listed as threatened under the ESA. In an important paper of 1994, York constructed a life table for SSL that indicated that a contributing factor of

K.L. Mashburn, S. Atkinson / General and Comparative Endocrinology 155 (2008) 352358

353

the decline of the Western stock was a decrease in juvenile survival that resulted in a chronic mortality rate of 1020% in that age class (York, 1994). The juvenile stage of life represents an important period of development for most organisms, both physically and socially. Physiologically, it is the time when most endocrine systems are being reactivated in such a manner that they will successfully ll the adult roles of reproduction and the rearing of ospring, as well as the ongoing requirement of maintaining homeostasis and nal stages of growth (Ebling, 2005; Gesquiere et al., 2005; Romeo, 2005). The failure of juvenile SSL to successfully complete this ontogenetic change may indicate an inability of the juvenile physiology to cope with both the normal changes required to attain adulthood and additional or extraordinary impacts within or upon their environment. The glucocorticoids, cortisol and corticosterone, secreted by the adrenal cortex in response to stimulation by adrenocorticotropic hormone (ACTH), play an integral role in an organisms ability to adapt to change. Glucocorticoids are major components of both metabolism (Zakrzewska et al., 1999; Khani and Tayek, 2001; Mastorakos and Zapanti, 2004) and stress responses (Carrasco and Van de Kar, 2003; Mostl and Palme, 2002). Both cortisol and fecal corticosteroids have been shown to be an accurate indicator of adrenal activity in response to both laboratory induced acute stress, as well as in response to predation, in adult SSLs. Mashburn and Atkinson (2004, 2007) illustrated that the adult SSLs were able to mount a classic stress response to exogenous ACTH, regardless of season, that could be accurately measured in serum as well as feces. Adult SSL also showed sex-dependant seasonal concentration dierences in fecal corticosteroids, likely due to reproductive and social inuences associated with the breeding season (Mashburn and Atkinson, 2007). The ability of the juvenile adrenals to exhibit a response to an acute stressor allows some insight into a physiology that, while undergoing a state of ux, is capable of supporting both the competing demands associated with ontogenetic change as well as transient perturbations to either their system or environment. In turn, denition of typical adrenal response in juveniles can aid in the establishment of physiological baselines for this age class of SSLs and may allow for more accurate identication of those individuals, or groups, that fall outside the norm. Leptin, a peptide hormone rst decribed in 1994, is produced primarily by adipocytes and is involved in the regulation of energy balance (Houseknecht et al., 1998; Casanueva and Dieguez, 1999). As receptors for this hormone are ubiquitous, it is speculated that leptin exerts eects on several dierent physiological systems and has been shown to function as a permissive factor in the attainment of puberty in laboratory studies in mice (Ahima et al., 1997; Chehab et al., 1997), rats (Chueng et al., 1997), and Rhesus monkeys (Suter et al., 2000; Plant, 2001). Though much of the research conducted to date continues to focus on the role of this hormone in the regulation of appetite as

a signal of satiety (Tang-Christensen et al., 1999; Morrison et al., 2001; Mustonen et al., 2005), it has been shown that the actions of glucocorticoids and leptin occur in complex association (Bornstein et al., 1997; Elimam et al., 1998; Pralong and Gaillard, 2001; Leal-Cerro et al., 2001). Marine mammals generally, and pinnipeds in particular, employ life history strategies that are heavily dependant on lipid reserves for both thermoregulation and as an energy source. The role of leptin, and its relationship with glucocorticoids, in these pinniped species may or may not be similar to that in terrestrial mammals. The purpose of this study is threefold: (1) to investigate the ability of juvenile SSL to respond to an acute stimulus, in the form of an ACTH challenge, under controlled conditions in the absence of human presence; (2) to investigate whether an elicited physiological response is similar between seasons but dierent than that measured in adult SSLs; and (3) to initiate investigation of the potentially complex relationship between the glucocorticoids and leptin in the developing juvenile in a species heavily reliant on lipid reserves for survival. The overall objective was to determine if the hypothalamic-pituitary-adrenal (HPA) axis in juvenile SSLs has reached the stage of development where the animal can adequately respond to the environmental and social dynamics of the reproductive season as well as challenges presented by maintaining growth and development during harsh environmental conditions.
2. Methods 2.1. Animals
Eight free-ranging reproductively intact juvenile Steller sea lions (6 males, 2 females; 1420 months) were captured at sea using the lasso technique reported by McAllister et al., 1997 o the Glacier Islands in Prince Wlliam Sound, Alaska. Ages were estimated by date of capture and through measurement of the canine tooth (Mellish et al., 2006; King et al., 2003). Animals TJ-009, TJ-0010, TJ-011, and TJ-012 were captured in November (TJ-010 the sole female in the group). Animals TJ-013, TJ0014, TJ-015, and TJ-016 were captured in June (TJ-014 the sole female of the group) (mean animal weight 157.8 6.5 kg, n = 8). Following an initial health screening, the animals were transported to the Alaska Sealife Center (ASLC, Seward, Alaska; N 6012 0 latitude, W 14942 0 longitude). These animals were temporarily held under ambient conditions at ASLC in a facility designed to limit both visual and aural contact with humans to enable the animals return to the wild after a three month period (Mellish et al., 2006). Each animal was housed separately, in individual pools, except during times at which animals were shifted to accommodate cleaning schedules. Three of the pools were 13 ft in diameter and one pool was 16 ft in diameter all four pools were 5 ft deep, and separated from each other by chain link fencing. Total haul out space available was 1600 sq. ft. and feeding occurred through a remote feeding system designed to introduce sh into each pool from a central location, without the sea lions associating food with humans.

2.2. ACTH challenge

Using protocols and materials found to be successful with adult SSLs (Mashburn and Atkinson, 2004, 2007), the juvenile SSLs were treated with a single injection (2 IU/kg, I.M.) of ACTH in a long-acting gel preparation (synthesized by Hadelds Pharmacy, Edmonds WA) in November 2004 and June 2005. Highly puried ACTH in a gel suspension provides

354

K.L. Mashburn, S. Atkinson / General and Comparative Endocrinology 155 (2008) 352358 Radioactivity of the bound portion was determined using a gamma counter (Gamma C12, Diagnostic Products Corporation, Los Angeles, CA). All samples from a single experiment were analyzed in duplicate within the same assay to reduce variation. Intra-assay coecients of variation were <5% and assay sensitivity was 0.5 ng/ml. Manufacturer crossreactivity data for the RIA are as follows: human leptin (100%), mouse leptin (73%), porcine leptin (67%), rat leptin (61%), canine leptin (3%) and not detectable for all other hormones tested.

sustained release of the ACTH and is typically used preferentially over aqueous-based preparations in studies with animals in which the detection of adrenal activity in the feces is desired (Wasser et al., 2000). Animals voluntarily entered transport cages and were moved to an indoor location out of visual and aural range of other individuals. Single injections (approximately 12 s in duration) occurred at approximately 09:00 and 13:00 on successive days (two animals each day for two days) and feces voided from each animal, obtained immediately prior to or during injection, were considered Time 0. Animals were anesthetized with isourane, a blood sample was drawn (Time 0) from the caudal plexus allowing the animals to serve as their own controls, followed immediately by ACTH injection. Blood samples (10 ml) were drawn every 15 min for up to 3 h via an indwelling catheter (18 gauge, 3 in.) placed in a hind-ipper vein. The catheter was ushed with approximately 3 ml sterile heparinized saline (concentration: 25 IU/ml) after each blood draw. At each new blood draw, the initial saline portion was discarded. After 3 h, animals were removed from isoourane. Once fully alert, animals were transferred to individual pools with no visual contact with human beings. All animals were monitored continuously via remote video cameras for 93 h post-ACTH administration, for a total of 96 h. Behaviors were recorded throughout this time and each defecation, when seen, was noted as to time and location for identication at collection. Every 6 h during the day, and as was part of standard husbandry practices, animals were rotated to adjacent enclosures and pools were drained of water. Both feces deposited on the deck and fecal material found in the bottom of the pool were collected. Animals were left undisturbed for 12 h overnight, and pools drained in the mornings to collected fecal material from the overnight period. All fecal material collected from the bottom of each pool was homogenized and subsampled. All fecal samples were stored at 20 C until processing and assay. To account for dierences in defecation times, fecal data were grouped into 4 h bins and are presented as means SEM for each of those bins.

2.6. Cortisol RIA

A solid-phase RIA kit (Diagnostic Products Corporation, Los Angeles, CA) previously validated for use in SSLs by Mashburn and Atkinson (2004) for cortisol was used to measure unextracted, undiluted Steller sea lion serum. All samples were analyzed in duplicate within the same assay to eliminate inter-assay variation, using methodology previously reported (Mashburn and Atkinson, 2004). Manufacturer cross-reactivity data are as previously reported in Mashburn and Atkinson (2007). Intra-assay coecients of variation were <5% and assay sensitivity was 4.8 ng/ml.

2.7. Corticosterone RIA

A double-antibody RIA kit (ICN Biomedicals Inc., Atascadero, CA) previously validated for use in Steller sea lions by Mashburn and Atkinson (2004) was used to measure fecal corticosteroids in extracted feces. Previous HPLC of juvenile fecal pools has indicated that, while a portion of the immunoreactivity measured by the RIA system is contributed by metabolites, the majority of immunoreactivity detected co-elutes with a 3Hlabeled corticosterone marker (Mashburn and Atkinson, 2004). All fecal samples were initially analyzed at a dilution of 1:4 in diluent buer provided by the manufacturer, using methodology previously reported by Mashburn and Atkinson (2004). Values from the RIA were corrected for dilution, extraction eciency, weight of fecal material extracted, and expressed as ng/g dry weight. Manufacturer cross-reactivity with other steroids were as previously reported in Mashburn and Atkinson (2007). Inter-assay coecients of variation for two separate assay controls were 9.07% (133.9 12.2 ng/ml) and 1.31% (696.9 19.14 ng/ml) for a low and a high control, respectively. Intra-assay coecients of variation were <5% and assay sensitivity was 13.7 ng/ml.

2.3. Blood samples

All sera used for this study, whether derived from whole blood collected via caudal plexus veinipuncture or through hind-ipper vein catheterization was collected into serum separator Vacutainers (Becton Dickinson, Franklin Lakes, NJ). All blood samples were refrigerated prior to serum harvest and 1 ml aliquots of sera were stored frozen at 80 C until radioimmunoassay (RIA).

2.8. Statistical analysis

Data are presented as means SEM unless otherwise noted. Comparisons of pre- and post-treatment data were analyzed using repeated measures analysis (one-way repeated measures ANOVA) with SigmaPlot 2002, v.8.0 (SPSS Inc. Chicago, IL).

2.4. Fecal sample extraction and preparation

Feces from individual juvenile Steller sea lions were fully mixed upon collection and treated as previously reported in Mashburn and Atkinson (2004). Sample dilutions were stored frozen at 20 C until RIA.

3. Results 3.1. Winter Radioimmunoassay of collected samples measured a 2.7fold increase in serum cortisol 90 min post-ACTH which was reected in a 16.7-fold increase in fecal corticosteroids 12 h post-ACTH at peak concentration in winter (Fig. 1). Fecal corticosteroid concentrations returned to baseline 32 h post-ACTH injection and the data indicated acute activity in juvenile adrenal glands is detectable in feces approximately 12 h post-ACTH, with a duration of approximately 20 h. Changes in fecal corticosteroid concentrations (baseline 71.8 13.7 ng/g; peak 1198.6 369.9 ng/g, F = 9.580; P = 0.053) did not prove statistically dierent in winter. Changes in serum cortisol concentrations proved statistically signicant (baseline 87.0 15.7 ng/ml; peak

2.5. Leptin RIA

A double-antibody RIA kit for leptin (Linco Research, St. Charles, MO) was modied and validated for use in undiluted, unextracted serum of male and female Steller sea lions. RIAs were run according to the manufacturers protocol with the following modications: all volumes were halved, an additional standard was added to the curve (i.e., one half the lowest standard) to increase sensitivity, the incubation period with primary antibody was doubled, and buer was not added to samples during incubation with the primary antibody. The modied system was validated for use with Steller sea lions using standard validation protocols as follows: Briey, serial dilutions of serum pools from both male and female Steller sea lions (neat to 1:8) yielded displacement parallel to that of the leptin standard curve. Recovery of added leptin (0.525 ng/ml) was 98.9% (SD = 3.2; CV = 3.3%) for males (y = 0.04 + 1.00x, r2 = 0.99) and 106.3% (SD = 15.6, CV = 14.7%) for females (y = 0.68 + 0.99x, r2 = 0.98). Intra-assay coecients of variation were <5% and assay sensitivity was 0.5 ng/ml.

K.L. Mashburn, S. Atkinson / General and Comparative Endocrinology 155 (2008) 352358

355

Fig. 1. Results of ACTH challenge, administered at time 0, in juvenile Steller sea lion feces in winter (n = 4). Fecal corticosterone ( ) exhibited a 16.7-fold increase at peak concentrations 12 h post-ACTH, a duration of 20 h, and returned to baseline by 32 h.

Fig. 3. Results of ACTH challenge, administered at time 0, in juvenile Steller sea lion feces in summer (n = 4). Fecal corticosterone ( ) exhibited a 10.4-fold increase at peak concentrations 28 h post-ACTH, a duration of 40 h, and returned to baseline by 52 h.

Fig. 2. Results of ACTH challenge, administered at Time 0, in juvenile Steller sea lion serum in winter (n = 4). Serum leptin ( ) reached peak concentrations by 45 min post-ACTH preceding serum cortisol ( ) peak concentrations by 45 min. Changes in both leptin and cortisol post-ACTH proved statistically signicant (P = 0.020 and 0.004, respectively).

Fig. 4. Results of ACTH challenge, administered at time 0, in juvenile Steller sea lion serum in summer (n = 4). Serum leptin ( ) reached peak concentrations by 45 min post-stimulus preceding serum cortisol ( ) concentration maxima by 105 min. Changes in cortisol, but not leptin, post-ACTH proved statistically signicant (P = 0.029 and 0.200, respectively).

237.55 10.0 ng/ml, F = 60.139; P = 0.004) and elevated concentrations were detected by 30 min post-ACTH. Changes in serum leptin (baseline 4.2 0.5 ng/ml; peak 7.5 0.6 ng/ml) proved to be statistically signicant (F = 16.60; P = 0.02) and peak concentrations preceded the serum cortisol peak by 45 min, returning to just above baseline concentrations 75 min post-ACTH (Fig. 2). 3.2. Summer Radioimmunoassay of collected samples measured a 3.2-fold increase in serum cortisol 90 min post-ACTH which was reected in 10.4-fold increase in fecal corticosteroids 28 h post-ACTH at peak concentrations in the summer (Fig. 3). Fecal corticosteroid concentrations returned to baseline 52 h post-ACTH and the data indicate acute activity in juvenile adrenal glands is detectable in feces approximately 1224 h post-ACTH, with a duration of approximately 40 h. Changes in fecal corticosteroids

proved signicant in summer (baseline 117.8 36.7 ng/g; peak 1219.3 298.4 ng/g, F = 12.515; P = 0.038). Elevated concentrations of serum cortisol were detected by 30 min post-ACTH (baseline 64.8 4.22 ng/ml; peak 209.5 18.0 ng/ml, F = 60.167; P = 0.004). Serum cortisol did not return to baseline concentrations in the summer for the duration of the sampling period. Changes in serum leptin (baseline 6.4 0.6 ng/ml; peak 18.7 6.9 ng/ml) were not statistically signicant (F = 3.363; P = 0.164) and preceded the serum cortisol concentration maxima by 105 min, returning to just above baseline concentrations 75 min post-ACTH (Fig. 4). 4. Discussion This study has demonstrated that ACTH elicits a response from juvenile Steller sea lion adrenal glands that can be measured in feces. This study also quanties the

356

K.L. Mashburn, S. Atkinson / General and Comparative Endocrinology 155 (2008) 352358

temporal context of onset of stimulus and output of corticosteroids in an acute stress response. There was considerable seasonal dierence in stimulus/response in juveniles, both between seasons and between individuals. The highest degree of individual variability occurred during the winter months, which is presumed to be the time when animals may incur the greatest metabolic challenges in the northern waters they inhabit. Juveniles in particular may face increased allostatic load (Korte et al., 2005; Landys et al., 2006) in the form of survival under the severe environmental conditions of sub-arctic winter coupled with the competing demand for physical growth and development. Thus, winter may be a critical period for animals of this age class as a period during which these animals depend most heavily on mounting an eective stress response to compensate for physical insult in times of presumed heaviest metabolic demands. Average age of sexual maturity for Steller sea lions is 3 years. The age of the animals in this study (1420 months), therefore, was dened as prepubertal. It was not the goal of this study to describe life stages in the species, but to begin to understand what may make this age class particularly vulnerable to environmental perturbations from a physiological perspective. Very little is understood regarding the endocrine physiology of the species because, to date, the techniques employed in the eld on free-ranging animals revealed little in the way of longitudinal information. The techniques used in this study have been successful in detecting seasonal dierences in adult Steller sea lion adrenal function, as well as measuring adrenal response of freeranging females to predation on pups in situ at Chiswell Island (Mashburn and Atkinson, 2007), and oers a unique means of investigating adrenal hormone changes in a longitudinal manner. In some ways, the juvenile response to ACTH as measured in feces was similar to that seen in the adults reported in previous studies (Mashburn and Atkinson, 2004, 2007). For instance, the duration of heightened adrenal activity in winter was approximately 20 h in juveniles and is reported as 20 h in the adults (Mashburn and Atkinson, 2004). The duration of increased fecal corticosteroids in summer ACTH trials was shown to be longer than that in winter in both juveniles (40 h, this study) and adults (36 h, Mashburn and Atkinson, 2007). Likewise, baseline concentrations of fecal corticosteroids in winter are similar in both the juveniles and the adults (71.8 13.7 ng/g and 88.5 28.9 juveniles and adults, respectively). However, juvenile animals did not exhibit the marked sex dierence seen in the corticosteroid concentrations reported in adults in summer. While this is not an unexpected result for reproductively immature animals, the limited number of females (1 each seasonal trial) may be a factor. Additionally in juveniles, the time to reach peak fecal concentrations in summer, while similar to that seen in the adults (28 h and 24 h, juveniles and adults, respectively) is considerably longer than the time to peak in winter

(12 h). This is in direct contrast to the adult SSLs, which attain peak fecal corticosteroid concentrations in winter 32 h post-ACTH, 8 h longer than is seen in summer. Peak adrenal response, as reected by fecal corticosteroids, not only occurs earlier in the winter but also returns to baseline 20 h earlier than in summer in juveniles. The winter juvenile peak also returns to baseline 20 h earlier than is exhibited by adults in winter. This implies that in juveniles, in winter, mechanisms both of initiation and cessation (or rate from start to nish) of the stress response are potentially more eective than in summer. However, the magnitude of the winter juvenile response is highly variable as is evidenced by the SEM which is over 30% that of the measured mean concentrations. This suggests that the magnitude of adrenal output of corticosteroids, and thus response to acute stress, is strongly individual in juvenile SSLs in the winter months. Presumably, winter presents greater environmental challenges to northern-dwelling marine mammals at a time during which it is imperative for an animal to cope with change in metabolic balance. This variability in adrenal response may indicate that juvenile animals respond by dierent magnitudes, though not at dierent rates, to physiological distress during times at which it may be most critical. Leptin has been investigated in a variety of terrestrial mammals. In contrast, very limited information regarding the role of leptin in marine mammals exists (Hammond et al., 2005; Arnould et al., 2002; Ortiz et al., 2001a, 2001b; Berman and Rea, 2000; Rea and Nagy, 2000). Marine mammals depend heavily on fat, or blubber, and are well adapted to a fat-based metabolism. However, conservation of blubber is essential to not only survival during natural periods of fasting, but thermogenesis and hydrodynamic eciency. Therefore, it is likely that there is a complex dynamic between leptin and the glucocorticoids particular to marine mammals that allows for successful growth and reproduction within a life history regime that typically involves extended periods of fasting. It is dicult to draw rm conclusions with regards to the interactions of the adrenals and leptin due to the use of a long-acting form of exogenous ACTH in this study. Normal feedback communication between leptin and the hypothalamus, and thus the hypothalamo-pituitary-adrenal axis (HPA), is circumvented by the continual release of ACTH into circulation. However, it is of interest to note that leptin concentrations reached peak values well in advance of cortisol and the leptin nadir concentrations appeared to be coincident with the peak concentrations of cortisol. Further, long-acting stimulation via ACTH appears to dampen further leptin activity which suggests that in a normally fed juvenile animal, both gluconeogenesis and lipolysis are enhanced through the glucocorticoid response while the leptin signal is abandoned. Patterns of secretion of leptin, as well as serum cortisol, also dier according to season, although it must be restated that the sustained release of the ACTH used most likely interferes with normal feedback loops following the most initial response.

K.L. Mashburn, S. Atkinson / General and Comparative Endocrinology 155 (2008) 352358

357

The current study has shown that there is a high degree of variability in juvenile SSLs, both in the response of leptin and fecal corticosteroids, to the introduction of a stressor in the form of a long-acting ACTH preparation. The variability appears to be associated with season and, in the case of corticosteroids, occurs in winter when a successful stress response is likely to be most critical. Further, it has been shown that juvenile SSL leptin responds more quickly than cortisol to a stressor in the form of ACTH stimulation, but that under continuous ACTH inuence leptin is suppressed. Thus, perturbations to the developing juvenile SSL, both acute and chronic, have the potential to disrupt the complex balance that must be maintained in the face of increased allostatic load in the form of competing demands of growth, development and thermoregulation in a species that utilizes adipose tissue for all of these. Additionally, the variability in adrenal response, its rapid onset, and termination may indicate that winter months represent a critical period in the juvenile SSLs and a time during which perturbations are likely to have a more pronounced eect on these animals. It is quite clear that further study is required to analyze both adrenal activity and the HPAleptin relationship in an age class of a species that appears to be particularly vulnerable. Many issues remain to be addressed with regards to both leptin and adrenal physiology in naturally obese animals, among them down-stream eects of changes in hormone concentrations. Logistical diculties in obtaining samples and lack of baseline information associated with Steller sea lions highlights the need for developing novel approaches to understanding the physiology of the species. Acknowledgements We thank Drs. JoAnn Mellish, Pam Tuomi and the ASLC husbandry, veterinary services and research departments for assistance with animal care and sampling; the ASLC dive/capture team, the captains and crew of the R/V Solstice, Auklet, and Stimson for assistance with animal capture and release; Don Calkins and Angie Steeves for program support. This project was supported by the ASLC Steller Sea Lion Research Program with funds from National Marine Fisheries Service. References
Ahima, R.S., Dushay, J., Flier, S.N., Prabakaran, D., Flier, J.S., 1997. Leptin accelerates the onset of puberty in normal female mice. J. Clin. Invest. 99, 391395. Arnould, J.P., Morris, M.J., Rawlins, D.R., Boyd, I.L., 2002. Variation in plasma leptin levels in response to fasting in Antarctic fur seals (Arctocephalus gazella). J. Comp. Physiol. B 172 (1), 2734. Berman, M., Rea, L. (2000). The eects of food dprivation on serum lipid concentration and content in Steller sea lions (Eumetopias jubatus). In: Proc. 3rd Comp. Nutr. Soc. Symp. Pacic Grove, CA, August 49, 2000, pp. 1316. Bickham, J.W., Patton, J.C., Loughlin, T.R., 1996. High variability for control region sequences in a marine mammal: implications for

conservation and biogeography of Steller sea lions. J. Mammol. 77, 95108. Bornstein, S.R., Uhlmann, K., Haidan, A., Ehrhart-Bornstein, M., Scherbaum, W.A., 1997. Evidence for a novel peripheral action of leptin as a metabolic signal to the adrenal gland: leptin inhibits cortisol release directly. Diabetes 46 (7), 12351238. Calkins, D.E., McAllister, D.C., Pitcher, K.W., 1999. Steller sea lion status and trend in Southeast Alaska. Mar. Mamm. Sci. 15 (2), 462 477. Carrasco, G.A., Van de Kar, L.D., 2003. The neuroendocrine pharmacology of stress. Eur. J. Pharmacol. 463 (13), 235272. Casanueva, F.F., Dieguez, C., 1999. Neuroendocrine regulation and actions of leptin. Front. Neuroendocrinol. 20, 317363. Chehab, F.F., Mounzih, K., Lu, R., Lim, M.E., 1997. Early onset of reproductive function in normal female mice treated with leptin. Science 275, 8890. Chueng, C.C., Thornton, J.E., Kuijper, J.L., Weigle, D.S., Clifton, D.K., Steiner, R.A., 1997. Leptin is a metabolic gate for the onset of puberty in the female rat. Endocrinology 138, 855858. Ebling, F.J.P., 2005. The neuroendocrine timing of puberty. Reproduction 129, 675683. Elimam, A., Knutsson, U., Bronnegard, M., Stierna, P., AlbertssonWikland, K., Marcus, C., 1998. Variations in glucocorticoid levels within the physiological range aect plasma leptin levels. Eur. J. Endocrinol. 139, 615620. Gesquiere, L.R., Altmann, J., Khan, M.Z., Couret, J., Yu, J.C., Endres, C.S., Lynch, J.W., Ogola, P., Fox, E.A., Alberts, S.C., Wango, E.O., 2005. Coming of age: steroid hormones of wild immature baboons (Papio cynocephalus). Am. J. Primatol. 67, 83100. Hammond, J.A., Bennet, K.A., Walton, M.J., Hall, A.J., 2005. Molecular cloning and expression of leptin in gray and harbor seal blubber, bone marrow, and lung and its potential role in marine mammal respiratory physiology. Am. J. Regul. Integr. Comp. Physiol. 289 (2), R545R553. Houseknecht, K.L., Baile, C.A., Matteri, R.L., Spurlock, M.E., 1998. The biology of leptin: a review. J. Anim. Sci. 76, 14051420. Khani, S., Tayek, J.A., 2001. Cortisol increases gluconeogenesis in humans: its role in the metabolic syndrome. Clin. Sci. 101, 739747. King, J.C., Gelatt, T.S., Pitcher, K.W., 2003. A eld based method for estimating age in free-ranging Steller sea lions. In: Proc. 15th Bienn. Conf. Mar. Mamm. Greensboro, NC, p. 201. Korte, S.M., Koolhaas, J.M., Wingeld, J.C., McEwen, B.S., 2005. The Darwinian concept of stress: benets of allostasis and costs of allostatic load and the trade-os in health and disease. Neuro. Biol. Rev. 29, 338. Landys, M.M., Ramenofsky, M., Wingeld, J.C., 2006. Actions of glucocorticoids at a seasonal baseline as compared to stress-related levels in the regulation of periodic life processes. Gen. Comp. Endo. 148, 132149. Leal-Cerro, A., Soto, A., Martinez, M.A., Dieguez, C., Casanueva, F.F., 2001. Inuence of cortisol status on leptin secretion. Pituitary 4, 111 116. Loughlin, T.A. (1997). Using the phylogenetic method to identify Steller sea lion stocks. In: Dizon, A.E, Chivers, S.J., Perrin, W.F. (Eds.) Molecular genetics of Marine Mammals. Special Publication No. 3. The Society for Marine Mammology, Lawernce, KS, pp. 159171. Loughlin, T.R., Perlov, A.S., Vladimirov, V.A., 1992. Range-wide survey and estimation of total numbers of Steller sea lions in 1989. Mar. Mamm. Sci. 8, 220239. Mashburn, K.L., Atkinson, S., 2004. Evaluation of adrenal function in serum and feces of Steller sea lions (Eumetopias jubatus): inuences of molt, gender, sample storage, and age on glucocorticoid metabolism. Gen. Comp. Endocrinol. 136 (3), 371381. Mashburn, K.L., Atkinson, S., 2007. Seasonal and predator inuences on adrenal function in adult Steller sea lions: gender matters. Gen. Comp. Endocrinol. 150, 246252. Mastorakos, G., Zapanti, E., 2004. The hypothalamic-pituitary-adrenal axis in the neuroendocrine regulation of food intake and obesity: the

358

K.L. Mashburn, S. Atkinson / General and Comparative Endocrinology 155 (2008) 352358 Rea, L.D., Nagy, T.R., 2000. Changes in serum leptin levels during fasting and food limitation in Steller sea lions (Eumetopias jubatus). In: Proc. 3rd Comp. Nutr. Soc. Symp. Pacic Grove, CA, August 49, 2000, pp. 171174. Romeo, R.D., 2005. Neuroendocrine and behavioral development during puberty: a tale of two axes. Vitam. Horm. 71, 125. Sease, J.L., Taylor, W.P., Loughlin, T.R., Pitcher, K.W., 2001. Aerial and land-based surveys of Steller sea lions (Eumetopias jubatus) in Alaska, June and July 1999 and 2000. U.S. Department of Commerce, NOAA Technical Memorandum NMFS-AFSC-122, 52 pp. Suter, K.J., Pohl, C.R., Wilson, M.E., 2000. Circulating concentrations of nocturnal leptin, growth hormone, and insulin-like growth factor-I increase before the onset of puberty in agonadal male monkeys: potential signals for the initiation of puberty. J. Clin. Endocrinol. Metab. 85, 808814. Tang-Christensen, M., Havel, P.J., Jacobs, R.R., Larsen, P.J., Cameron, J.L., 1999. Central administration of leptin inhibits food intake, and activates the sympathetic nervous system in rhesus macaques. J. Clin. Endocrinol. Metab. 84, 711717. Wasser, S.K., Hunt, K.E., Brown, J.L., Cooper, K., Crockett, C.H., Bechrt, U., Millspaugh, J.J., Larson, S., Monfort, S.L., 2000. A generalized fecal glucocorticoid assay for use in a diverse array of nondomestic mammalian and avian species. Gen. Comp. Endo. 120, 260 275. York, A., 1994. The population dynamics of Northern sea lions 1975 1985. Mar. Mamm. Sci. 10 (1), 3851. Zakrzewska, K.E., Cusin, I., Stricker-Kongrad, A., Boss, O., Ricquier, D., Jeanrenaud, B., Rohner-Jeanrenaud, F., 1999. Induction of obesity and hyperleptinemia by central glucocorticoid infusion in the rat. Diabetes 48, 365370.

role of corticotrophin releasing hormone. Nutr. Neurosci. 7 (56), 271 280. McAllister, D., Calkins, D., Cunningham, W., 1997. Steller sea lion capture techniques development. In: Pitcher, K. (Ed.), Steller sea lion recovery investigations in Alaska, 19951996. Alaska Department of Fish and Game, Anchorage, Alaska, pp. 8689, 89 pp. Mellish, J.E., Calkins, D.G., Christen, D.R., Horning, M., Rea, L.D., Atkinson, S.K., 2006. Temporary captivity as a research tool: comprehensive study of wild pinnipeds under controlled conditions. Aqu. Mamm. 32 (1), 5865. Morrison, C.D., Daniel, J.A., Holmberg, B.J., Djiane, J., Raver, N., Gertler, A., Keisler, D.H., 2001. Central infusion of leptin into wellfed, and undernourished ewe lambs: eects on feed intake, and serum concentrations of growth hormone, and luteinizing hormone. J. Endocrinol. 163, 317324. Mostl, E., Palme, R., 2002. Hormones as indicators of stress. Domest. Anim. Endocrinol. 23 (12), 6774. Mustonen, A., Pyykonen, T., Asikainen, J., Hanninen, S., Mononen, J., Nieminen, P., 2005. Circannual leptin and ghrelin levels of the blue fox (Alopex lagopus) in reference to seasonal rhythms of body mass, adiposity and food intake. J. Exp. Zool. A 303, 2636. Ortiz, R.M., Noren, D.P., Litz, B., Ortiz, C.L., 2001a. A new perspective on adiposity in a naturally obese mammal. Am. J. Physiol. Endocrinol. Metab. 281 (6), E1347E1351. Ortiz, R.M., Wade, C.E., Ortiz, C.L., 2001b. Eects of prolonged fasting on plasma cortisol and TH in postweaned northern elephant seal pups. Am. J. Regul. Integr. Comp. Physiol. 280, R790R795. Plant, T.M., 2001. Leptin, growth hormone, and the onset of primate puberty. J. Clin. Endocrinol. Metab. 86, 14591460. Pralong, F.P., Gaillard, R.C., 2001. Neuroendocrine eects of leptin. Pituitary 4, 2532.