Department of Biology, University of California, Santa Cruz, Santa Cruz, California 95064
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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: 43.9 ± 0.5, late: 45.5 ± 0.5%) and leptin [early: 2.9 ± 0.1 ng/ml human equivalents (HE), late: 3.0 ± 0.1 ng/ml HE] did not change. Cortisol increased between early (9.2 ± 0.5 µg/dl) and late (16.3 ± 0.9 µg/dl) periods and was significantly and negatively correlated with BM (r = 0.426; P < 0.0001) 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
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
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
(3H2O) 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 3H2O 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 3H2O. An equilibration
blood sample was taken 3 h after dosing. Estimations of TBW and FM
by 3H2O 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 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 intra-assay 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 < 0.05. Statistical analyses were made using Statview (16).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 = 125.4 1.5 cortisol; r = 0.384; P = 0.0005)
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 = 56
0.64 cortisol;
r = 0.288; P < 0.0001).
|
|
|
|
![]() |
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 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.
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.
![]() |
ACKNOWLEDGEMENTS |
---|
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.
![]() |
FOOTNOTES |
---|
This research was supported by Minority Access to Research Careers Grant GM-58903-01 (C. L. Ortiz), NASA the Graduate Student Research Program NGT-2-52230 (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. Ortiz.
Address for reprint requests and other correspondence: R. M. Ortiz, (E-mail: rudy{at}biology.ucsc.edu).
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.
Received 29 March 2001; accepted in final form 22 August 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ahima, RS,
Prabakaran D,
Mantzoros C,
Qu D,
Lowell B,
Maratos-Flier E,
and
Flier JS.
Role of leptin in the neuroendocrine response to fasting.
Nature
3382:
250-252,
1996.
2.
Bergendahl, M,
Vance ML,
Iranmanesh A,
Thorner MO,
and
Veldhuis JD.
Fasting as a metabolic stress paradigm selectively amplifies cortisol secretory burst mass and delays the time of maximal nyctohemeral cortisol concentrations in healthy men.
J Clin Endocrinol Metab
81:
692-699,
1996[Abstract].
3.
Brillon, DJ,
Zheng B,
Campbell RG,
and
Matthews DE.
Effect of cortisol on energy expenditure and amino acid metabolism in humans.
Am J Physiol Endocrinol Metab
268:
E501-E513,
1995
4.
Caro, JF,
Sinha MK,
Kolaczynski JW,
Zhang PL,
and
Considine RV.
Leptin: the tale of an obesity gene.
Diabetes
45:
1455-1462,
1996[ISI][Medline].
5.
Castellini, MA,
Costa DP,
and
Huntley AC.
Fatty acid metabolism in fasting elephant seal pups.
J Comp Physiol B
157:
445-449,
1987[ISI][Medline].
6.
Considine, RV,
and
Caro JF.
Leptin and the regulation of body weight.
Int J Biochem Cell Biol
29:
1255-1272,
1997[ISI][Medline].
7.
Considine, RV,
Sinha MK,
Heiman ML,
Kriauciunas A,
Stephens TW,
Nyce MR,
Ohannesian JP,
Marco CC,
McKee LJ,
Bauer TL,
and
Caro JF.
Serum immunoreactive-leptin concentrations in normal-weight and obese humans.
N Engl J Med
334:
292-295,
1996
8.
Green, PK,
Wilkinson CW,
and
Woods SC.
Intraventricular corticosterone increases the rate of body weight gain in underweight adrenalectomized rats.
Endocrinology
130:
269-275,
1992[Abstract].
9.
Hickey, MS,
Considine RV,
Israel RG,
Mahar TL,
McCammon MR,
Tyndall GL,
Houmard JA,
and
Caro JF.
Leptin is related to body fat content in male distance runners.
Am J Physiol Endocrinol Metab
271:
E938-E940,
1996
10.
Maffei, M,
Halaas J,
Ravussin E,
Pratley RE,
Lee GH,
Zhang Y,
Fei H,
Kim S,
Lailone R,
Ranganathan S,
Kern PA,
and
Friedman JM.
Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects.
Nature Med
1:
1155-1161,
1995[ISI][Medline].
11.
Ortiz, CL,
Costa D,
and
Le Boeuf BJ.
Water and energy flux in elephant seal pups fasting under natural conditions.
Physiol Zool
51:
166-178,
1978[ISI].
12.
Ortiz, RM,
Wade CE,
and
Ortiz CL.
Effects of prolonged fasting on plasma cortisol and TH in postweaned northern elephant seal pups.
Am J Physiol Regulatory Integrative Comp Physiol
280:
R790-R795,
2001
13.
Pasman, WJ,
Westerterp-Plantenga MS,
and
Saris WHM
The effect of exercise training on leptin levels in obese males.
Am J Physiol Endocrinol Metab
274:
E280-E286,
1998
14.
Rea, LD,
and
Costa DP.
Changes in standard metabolism during long-term fasting in northern elephant seal pups (Mirounga angustirostris).
Physiol Zool
65:
97-111,
1992[ISI].
15.
Samuels, MH,
and
McDaniel PA.
Thyrotropin levels during hydrocortisone infusions that mimic fasting-induced cortisol elevations: a clinical research center study.
J Clin Endocrinol Metab
82:
3700-3704,
1997
16.
SAS Institute. Statview. Cary, NC: SAS Institute, 1998.
17.
Stephens, TW,
and
Caro JF.
To be lean or not to be lean. Is leptin the answer?
Exp Clin Endocrinol Diabetes
106:
1-15,
1998[ISI][Medline].
18.
Tataranni, PA,
Larson DE,
Snitker S,
Young JB,
Flatt JP,
and
Ravussin E.
Effects of glucocorticoids on energy metabolism and food intake in humans.
Am J Physiol Endocrinol Metab
271:
E317-E325,
1996
19.
Whittow, GC.
Thermoregulatory adaptations in marine mammals: interacting effects of exercise and body mass: a review.
Mar Mamm Sci
3:
220-241,
1987[ISI].
20.
Worthy, GAJ,
and
Lavigne DM.
Energetics of fasting and subsequent growth in weaned harp seal pups, Phoca groenlandica.
Can J Zool
61:
447-456,
1983[ISI].
21.
Young, RA.
Fat, energy and mammalian survival.
Amer Zool
16:
699-710,
1976[ISI].