RUDY M. ORTIZ, DAWN P. NOREN, BEATE LITZ, AND C. LEO ORTIZ Department of Biology, University of California, Santa Cruz, Santa Cruz, California 95064

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1 Am J Physiol Endocrinol Metab 281: E1347 E1351, A new perspective on adiposity in a naturally obese mammal rapid communication RUDY M. ORTIZ, DAWN P. NOREN, BEATE LITZ, AND C. LEO ORTIZ Department of Biology, University of California, Santa Cruz, Santa Cruz, California Received 29 March 2001; accepted in final form 22 August 2001 Ortiz, Rudy M., Dawn P. Noren, Beate Litz, and C. Leo Ortiz. A new perspective on adiposity in a naturally obese mammal. Am J Physiol Endocrinol Metab 281: E1347 E1351, Many mammals seasonally reduce body fat due to inherent periods of fasting, which is associated with decreased leptin concentrations. However, no data exist on the correlation between fat mass (FM) and circulating leptin in marine mammals, which have evolved large fat stores as part of their adaptation to periods of prolonged fasting. Therefore, FM was estimated (by tritiated water dilution), and serum leptin and cortisol were measured in 40 northern elephant seal (Mirounga angustirostris) pups early ( 1 wk postweaning) and late (6 8 wk postweaning) during their natural, postweaning fast. Body mass (BM) and FM were reduced late; however, percent FM (early: , late: %) and leptin [early: ng/ml human equivalents (HE), late: ng/ml HE] did not change. Cortisol increased between early ( g/dl) and late ( g/dl) periods and was significantly and negatively correlated with BM (r 0.426; P ) and FM (r 0.328; P 0.003). FM and percent FM were not correlated (P 0.10) with leptin at either period. The present study suggests that these naturally obese mammals appear to possess a novel cascade for regulating body fat that includes cortisol. The lack of a correlation between leptin and FM may reflect the different functions of fat between terrestrial and marine mammals. body composition; cortisol; fat mass; obesity; pinnipeds Address for reprint requests and other correspondence: R. M. Ortiz, ( rudy@biology.ucsc.edu). CIRCULATING LEPTIN CONCENTRATIONS have been positively correlated with fat mass (FM) in humans and rodents, thereby providing an index of adiposity (4, 9, 10, 13). For example, lean humans with 10% body fat may exhibit leptin concentrations of 2 ng/ml (9), whereas obese humans with 30% body fat may exhibit leptin concentrations greater than 10 ng/ml (7, 13). Therefore, mammals with relatively large percent FM would be expected to exhibit relatively high circulating leptin concentrations. Marine mammals have evolved large body fat stores as an adaptation for inhabiting a marine environment (19, 21) and for fasting for extended periods (11). For example, northern elephant seal (NES; Mirounga angustirostris) pups exhibit percent FM of 50% after nursing (11, 14), which would be expected to be associated with relatively higher leptin concentrations than in those mammals previously studied. However, our previous data showed that leptin concentrations were relatively low and similar to those of lean mice (12), suggesting that leptin may not play a significant role in the regulation of body fat in these animals. Alternatively, we showed that plasma cortisol concentrations increased linearly over the course of the fast and may parallel the decrease in fat mass over the same time (14), suggesting that glucocorticoids may be associated with the regulation of body fat in these mammals. The involvement of glucocorticoids in adiposity, appetite, and energetics in humans has been well examined (3, 18). FM and circulating leptin and cortisol in any marine mammal have yet to be correlated, which should provide a better understanding of the role that leptin and cortisol play in regulating body fat in these animals. Therefore, NES pups were used as a model to examine the association between estimated body fat and serum leptin and cortisol concentrations to determine whether these parameters are correlated in a naturally obese mammal. The present study also reports on leptin concentrations in a larger sample size (40 vs. 15 animals) and examines the relationships between circulating cortisol and leptin concentrations with body fat, all of which extend the findings of our previous study (12). METHODS All methods were reviewed and approved by University of California Santa Cruz Chancellor s Animal Research Committee. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact /01 $5.00 Copyright 2001 the American Physiological Society E1347

2 E1348 LEPTIN IN A FAT MAMMAL Animals. Forty NES pups (20 males, 20 females) from Año Nuevo State Reserve ( 30 km north of Santa Cruz, CA) were studied. Body mass (BM) and blood samples were obtained, and FM was estimated early ( 1 wk postweaning) and late (6 8 wk postweaning) during the pups natural, postweaning fast. Animals were left in their natural habitat between sampling periods. Mother-pup pairs were individually identified to determine the date of weaning. A pup was considered weaned when its mother was not seen on the subsequent day. Procedures and analyses. Procedures were similar at both the early and late sampling periods. BM was measured using a hanging-load cell suspended from a tripod. After weighing, pups were sedated with 0.01 ml tiletamine HCl and zolazepam HCl (Telazol; Fort Dodge Animal Health, Fort Dodge, IA)/kg BM so that a 16-gauge, 3.5-in. spinal needle could be inserted into the extradural spinal vein, from which a blood sample was obtained and tritium ( 3 H 2O) was infused. Blood (11 ml) was collected into an untreated vacutainer tube. After the predose blood sample was collected, pups received 0.3 mci of 3 H 2O in 3 ml of sterile water to estimate total body water (TBW) and FM. The infusion needle was flushed once with 5 ml of sterile saline to ensure complete delivery of 3 H 2O. An equilibration blood sample was taken 3 h after dosing. Estimations of TBW and FM by 3 H 2O dilution were calculated as previously described (11). After collection, blood tubes were placed on ice in a portable ice chest until they could be returned to the lab to be centrifuged, which was within 6 h. Blood samples were then centrifuged for 15 min (1,500 g at 4 C), and serum was collected and frozen at 70 C for later analyses. Immunoreactive leptin (multispecies kit, Linco, St. Charles, MO) and cortisol (DPC, Los Angeles, CA) were assayed from the predose blood sample by means of commercially available radioimmunoassay kits previously validated for NES plasma (12). A representative estimation of the percentage of cross-reactivity between immunoreactive NES leptin and human leptin antibody is displayed in Fig. 1. Because the antibody was raised against human leptin and human leptin standards were used, immunoreactive NES leptin concentrations are expressed in nanograms per milliliter human equivalents (ng/ml HE). The Fig. 2. Correlation between body mass and fat mass (FM) in NES pups (n 40) measured early ( 1 wk postweaning) and late (6 8 wk postweaning) during their postweaning fast. Correlation was considered significant at P minimum detectable concentration is 1 ng/ml HE as reported by the manufacturer. Percent recovery of exogenous human leptin from pooled NES plasma was 92%. All samples were analyzed in duplicate and run in a single assay with intraassay percent coefficient of variability 9% for both assays. We recognize that the leptin bound in the current assay is solely immunoreactive; however, for the sake of brevity, we will refer to this molecule as simply leptin for the remainder of the paper. Statistics. Means ( SE) were compared by paired t-test between early and late sampling periods. Correlations between FM and percent FM with circulating leptin and cortisol were determined by simple regression. Differences were considered significant at P Statistical analyses were made using Statview (16). RESULTS Fig. 1. Representative estimation of the percentage of cross-reactivity between northern elephant seal (NES) plasma and human leptin antibody. HE, human equivalent; B/Bo, percent binding. BM, FM, and percent FM did not exhibit any gender differences (P 0.10) at either sampling period; therefore, these data were pooled. BM and FM exhibited a significant and positive correlation between early and late fasting periods (Fig. 2). The decrease in FM accounted for 40 1% of the decrease in BM (Table 1). With a hydration state of muscle of 73% (D. P. Noren, unpublished data), water accounted for 44 1% of the decrease in BM, and thus protein was responsible for the remaining 16%. Leptin and percent FM were not significantly altered (Table 1). FM and percent FM were not significantly correlated with leptin (Fig. 3). Cortisol increased significantly between early and late periods (Table 1) and exhibited a significant and negative correlation with BM (BM cortisol; r 0.384; P ) and FM (Fig. 4), but no relationship with percent FM. Although an obvious outlier is present in the correlation between cortisol and FM, the relationship remains significant after this value is removed (FM cortisol; r 0.288; P ).

3 LEPTIN IN A FAT MAMMAL E1349 Table 1. Body mass, fat mass, percent fat mass, and serum leptin and cortisol concentrations from 40 northern elephant seal pups sampled early and late during their natural postweaning fast Early ( 1 wk) Late (6 8 wk) Significance Body mass, kg Fat mass, kg Fat mass, % NS Leptin, ng/ml HE NS Cortisol, g/dl Values are means SE. HE, human equivalent. Means were compared by paired t-test and considered significant at P NS, not significant. DISCUSSION Fasting is a natural component of the life history of many terrestrial and marine mammals and is usually associated with a decrease in both body fat (21) and circulating leptin (1). However, in the present study, the reduction in body fat during the 7-wk fasting period was independent of a concomitant decrease in circulating leptin but was negatively correlated with serum cortisol. The relationships of cortisol with BM and FM Fig. 3. Relationship between FM and serum leptin (A) and %FM and serum leptin concentrations (B) in 40 NES pups sampled early and late during their natural, postweaning fast. Neither correlation was significant (P 0.10). Fig. 4. Relationship between serum cortisol concentrations and FM in 40 NES pups sampled early and late during their natural, postweaning fast. Correlations were considered significant at P suggest that cortisol may be involved in the regulation of adiposity in these naturally obese mammals. Although fasting is usually associated with a reduction in BM and thus serum leptin concentrations (1), leptin concentrations were not reduced between the early and late fasting periods in the present study, suggesting that leptin is dissociated from the reductions in BM and FM during the fast in NES pups and that it may not play a role in regulating FM. Alternatively, the lack of a further reduction in leptin concentration between the early and late fasting periods may be attributed to the possibility that leptin concentrations were maximally reduced before the initial measurement. In humans and rodents, maximal reduction in circulating leptin can be achieved within 24 h postfasting with these reduced levels independent of body fat content (1, 4). Regardless, alternative mechanisms may exist that regulate BM, and thus body fat, during this period in these and other marine mammals. These mechanisms may include the involvement of glucocorticoids, as suggested by the negative correlations between cortisol and BM and FM in the present study. Forced-fasting in mammals is usually associated with an elevation in glucocorticoids (2, 15), which induce lipolysis, providing substrates for subsequent metabolism necessary to maintain the animals during periods of food deprivation. The significant, although weak, correlations between cortisol and BM and FM in the present study suggest that this glucocorticoid may contribute significantly to the reported increase in fat oxidation during the fast (5, 14). Also, glucocorticoids have been shown to promote feeding behavior (8); therefore, the elevated cortisol concentrations may serve as a cue to terminate fasting and initiate feeding. Thus increased cortisol-induced lipolysis may result in FM reaching a critical lower limit at the end of the fast and thereby stimulate the pups to depart from the rookery and beginning foraging.

4 E1350 LEPTIN IN A FAT MAMMAL In humans and rodents, circulating leptin concentrations correlate well with body fat, thus usually providing a reliable index of FM (4, 7, 9, 10, 13). However, the present study did not demonstrate a correlation between circulating leptin concentrations and FM or percent FM in these naturally obese mammals. The lack of a significant and positive relationship between leptin and body fat in NES pups suggests that leptin may not play a significant role in the regulation of FM in these animals and thus does not provide a reliable index of body fat as it does in terrestrial mammals. The lack of a correlation between FM and circulating leptin may be attributed to the possibility that leptin concentrations had already been maximally reduced due to fasting, as previously mentioned. However, leptin concentrations can exhibit a large degree of variation for any given percent body fat, even though they usually parallel the degree of adiposity in mammals (17). These conditions have been described as hyperleptinema (or leptin resistance) and hypoleptinema (or leptin deficiency) (17). Pups in the present study were 45% body fat, yet circulating leptin concentrations were relatively low and similar to those for lean humans (9) and mice (1), suggesting that these animals may be inherently leptin deficient. Under nonfasting conditions, these animals may continue to accrue body fat independently of leptin s inhibitory actions on food intake (6, 17), which is essential for their survival in a marine environment. Therefore, a condition of chronic hypoleptinema would behoove marine mammals, although this condition could be associated with some pathologies in humans (17). The lack of a correlation between leptin and FM may also reflect the difference in functions of fat depots between terrestrial and marine mammals. Marine mammals evolved large fat depots primarily associated with the blubber layer (composed predominantly of white fat and accounting for 94% of body fat) (20) as an adaptation to an aquatic environment (19, 21) and to prolonged fasting (11). Blubber fat is the primary source of energy during prolonged fasting (11) and is essential for insulation from cold marine waters (19, 21), neither of which conditions is commonly observed in terrestrial mammals. In fasting NES pups, unlike other mammals, the results of the present study suggest that cortisol, and not leptin, plays a significant role in the regulation of BM and fat. Cortisol may affect body fat by increasing lipolysis to a point that a critical lower BM is reached and thus stimulate the pups to terminate the fast and initiate foraging. Relative hypoleptinema may play a role in the development of obesity (17), for which NES pups may provide an interesting study model. We thank the many student volunteers from UCSC for helping in the field, and G. Gurun, B. Litz, and A. Ramirez for their assistance in the lab. We thank G. Strachan and all the rangers at Año Nuevo for allowing us access to the animals and for their assistance in the field. We also thank Drs. J. G. Mercer and C. E. Wade for their comments on the manuscripts. This research was supported by Minority Access to Research Careers Grant GM (C. L. Ortiz), NASA the Graduate Student Research Program NGT (R. M. Ortiz), California Space Grant Consortium (R. M. Ortiz), Grant-in-Aid of Research from Sigma Xi (R. M. Ortiz), American Museum of Natural History (D. P. Noren and R. M. Ortiz), Dr. Earl H. Myers and Ethel M. Myers Marine Biology Trust (D. P. Noren and R. M. Ortiz), Friends of Long Marine Lab (D. P. Noren and R. M. Ortiz), and University of California Natural Reserve System (D. P. Noren). Research was conducted under National Marine Fisheries Service marine mammal permit no. 836 to C. L. 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