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Serum corticosteroid binding globulin expression is modulated by fasting in polar bears (Ursus maritimus)
Comparative Biochemistry and Physiology, Part A 158 (2011) 111–115
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Comparative Biochemistry and Physiology, Part A j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a
Serum corticosteroid binding globulin expression is modulated by fasting in polar bears (Ursus maritimus) Brian A. Chow a, Jason Hamilton a, Marc R.L. Cattet b, Gordon Stenhouse c, Martyn E. Obbard d, Mathilakath M. Vijayan a,⁎ a
Department of Biology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada Canadian Cooperative Wildlife Health Centre, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5B4, Canada Foothills Research Institute, Hinton, Alberta, T7V 1X7, Canada d Wildlife Research and Development Section, Ontario Ministry of Natural Resources, Trent University, Peterborough, Ontario, K9J 7B8, Canada b c
a r t i c l e
i n f o
Article history: Received 11 May 2010 Received in revised form 16 September 2010 Accepted 20 September 2010 Available online 29 September 2010 Keywords: CBG Cortisol Fasting Protein expression Stress response Ursids
a b s t r a c t Polar bears (Ursus maritimus) from several subpopulations undergo extended fasting during the ice-free season. However, the animals appear to conserve protein despite the prolonged fasting, though the mechanisms involved are poorly understood. We hypothesized that elevated concentrations of corticosteroid binding globulin (CBG), the primary cortisol binding protein in circulation, lead to cortisol resistance and provide a mechanism for protein conservation during extended fasting. The metabolic state (feeding vs. fasting) of 16 field sampled male polar bears was determined based on their serum urea to creatinine ratio (N 25 for feeding vs. b 5 for fasting). There were no significant differences in serum cortisol levels between all male and female polar bears sampled. Serum CBG expression was greater in lactating females relative to nonlactating females and males. CBG expression was significantly higher in fasting males when compared to nonfasting males. This leads us to suggest that CBG expression may serve as a mechanism to conserve protein during extended fasting in polar bears by reducing systemic free cortisol concentrations. This was further supported by a lower serum glucose concentration in the fasting bears. As well, a lack of an enhanced adrenocortical response to acute capture stress supports our hypothesis that chronic hunger is not a stressor in this species. Overall, our results suggest that elevated serum CBG expression may be an important adaptation to spare proteins by limiting cortisol bioavailability during extended fasting in polar bears. © 2010 Elsevier Inc. All rights reserved.
1. Introduction The polar bear (Ursus maritimus) is a large arctic carnivore that occupies the top trophic level in a marine food web (Hobson and Welch, 1992). In the Canadian arctic, they primarily feed on seals (Phocidae) and rely on sea ice as a seasonal platform upon which they hunt their prey. During the ice-free period (Jun–Oct), populations are forced ashore or onto unproductive multi-year ice, where bears generally undergo extended fasting (Stirling and Derocher, 1993). Although polar bears have been observed to forage while on land, it is generally accepted that these supplemental food sources do not contribute significantly to their diet (Lunn and Stirling, 1985). As year-round opportunistic feeders, polar bears are known to enter a facultative hypometabolic state that is similar to hibernation in American black (U. americanus) and grizzly bears (U. arctos) during periods of sparse food availability, as evidenced by the lowering of heart rate, body temperature, and serum urea to creatinine (U/C) ratio
⁎ Corresponding author. Tel.: + 1 519 888 4567x32035; fax: + 1 519 746 0614. E-mail address: [email protected] (M.M. Vijayan). 1095-6433/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2010.09.017
(Hellgren, 1998; Ramsay et al., 1991). However, unlike these species, most polar bears may remain in an active, “walking hibernation” state while fasting. An exception is pregnant females, which will enter maternity dens to give birth and initiate lactation during this period (Nelson et al., 1983). Corticosteroids are thought to play an important role during extended fasting in animals by mobilizing energy substrates, including amino acids from proteins to fuel metabolic processes critical for survival (Schwartz et al., 1995). Corticosteroids are secreted from the adrenal cortex in response to stressor-induced activation of the hypothalamus–pituitary–adrenal (HPA) axis. The primary step involves the release of corticotropin releasing hormone from the hypothalamus in response to sensory neural inputs, stimulating the pituitary gland to release adrenocorticotrophic hormone. This peptide then activates melanocortin 2 receptors on the adrenal cortex, leading to cortisol biosynthesis (Sapolsky et al., 2000). Elevation in serum corticosteroid levels stimulates muscle protein catabolism and enhances gluconeogenesis especially from amino acids in the liver, resulting in hyperglycemia (Brillon et al., 1995; Mommsen et al., 1999). However, little is known about the role of cortisol in the metabolic adaptation of bears to extended fasting.
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It is known that the levels of serum total cortisol, the principal corticosteroid in bears, are elevated in denning black bears relative to summer active bears (Harlow et al., 1990). However, despite hypercortisolemia, black bears conserve their lean (protein) body mass during denning (Hellgren, 1998; Lohuis et al., 2007), leading us to hypothesize that these animals may be resistant to corticosteroid action during extended fasting. Studies have shown that in mammals up to 95% of circulating corticosteroid is bound to a 55 kDa glycoprotein, corticosteroid-binding globulin (CBG) (Gayrard et al., 1996; Rosner, 1990). The binding of the steroid to CBG reduces the free cortisol concentration, which is the biologically active fraction. Consequently, serum CBG concentrations play a major role in regulating corticosteroid bioavailability and target tissue responses (Rosner, 1990). Against this background, we tested the hypothesis that serum CBG expression is higher in fasting polar bears as an adaptation to limit cortisol bioavailability and protein catabolism. In addition, we compared serum CBG expression in adult male and female polar bears to explore differences associated with sexual dimorphism and confirm previous findings in other mammals (Mataradze et al., 1992; Fernandez-Real et al., 2002; Chow et al., 2010). 2. Materials and methods 2.1. Polar bear serum samples We obtained archived frozen serum samples (2–4 mL per bear) from 40 polar bears (n = 24 males, 16 females) that had been captured and released in conjunction with long-term research projects in the Canadian Arctic conducted by the University of Saskatchewan from 1989 to 1997, and the Ontario Ministry of Natural Resources from 2002 to 2009. All bears were located and immobilized from a helicopter by remote drug delivery (Palmer Cap-Chur Inc., Powder Springs, GA, USA) as detailed in Cattet et al. (1997) and Cattet and Obbard (2010). Samples were chilled on icepacks in a cooler immediately following blood collection to slow down blood cell metabolism. All samples were collected and processed similarly, with sera frozen (−25 °C) within 8 h of collection in long-term storage at the University of Saskatchewan. The research protocols for both projects were approved annually by animal care committees at the University of Saskatchewan and Ontario Ministry of Natural Resources and was in accordance with guidelines provided by the Canadian Council on Animal Care (2003) for the safe handling of wildlife. 2.2. Serum CBG expression 2.2.1. Comparison by sex To evaluate sexual dimorphism in CBG expression in polar bears, we compared levels in samples collected from 24 polar bears captured from the area of southern Hudson Bay during the months of September and October. All samples were from adult (≥5 years) bears of similar body condition within three sex and reproductive classes: lactating females accompanied by dependent offspring (“lactating females”, n = 8), non-lactating solitary females (“solitary females”, n = 8), and males (n = 8). 2.2.2. Comparison by metabolic state To compare CBG levels between feeding and fasting states, samples were collected from 16 adult (≥5 years) male polar bears. Eight animals were captured on sea ice during April and May, a time when most bears are in a feeding state, from the western Hudson Bay (WHB) and Lancaster Sound (LS) subpopulations. Another eight animals were captured on land from the WHB subpopulation during October, when most polar bears are in a fasting state. Serum urea to creatinine (U/C) ratios have been shown to be a reliable marker of metabolic state in polar bears (Ramsay et al., 1991). We determined serum urea and creatinine concentrations using an Abbott Spectrum H
Series II biochemistry analyzer (Abbott Laboratories Diagnostic Division, Abbott Park, IL, USA) to confirm the metabolic state of bears at the time of capture.
2.2.3. Validation of CBG cross-reactivity in polar bears A peptide region of a previously sequenced grizzly bear CBG was used to generate an affinity purified rabbit polyclonal antibody, which was then used to test for cross-reactivity in polar bears as previously described (Chow et al., 2010). Specifically, polar bear serum was depleted of albumin and IgG with Aurum Serum Protein Mini-kits (Bio-Rad, Hercules, CA, USA) and total protein concentration was determined by the bicinchoninic acid method (Smith et al., 1985) using bovine serum albumin as the standard. Samples were serially diluted to 2.5, 5, 10, and 20 μg total serum protein in sample buffer [0.06 M Tris-HCl (pH 6.8), 25% (v/v) glycerol, 0.02% (w/v) SDS, 0.001% (w/v) bromophenol blue (Fisher Scientific, Fair Lawn, NJ, USA)] and loaded into wells on 10% reducing polyacrylamide gels for immunodetection. A low-range molecular weight marker (Bio-Rad) was used to confirm molecular mass of the protein. Proteins were separated (200 V for 50 min; Mini Protean III, Bio-Rad) using a discontinuous buffer (Laemmli, 1970). The separated proteins were transferred to a 0.22 μm pore size nitrocellulose membrane using a Transblot SD SemiDry Electrophoretic Transfer Cell (Bio-Rad). Equal loading and transfer efficiency were inspected by Ponceau S (Bio-Rad) staining of the membrane. The membranes were rinsed and blocked with blocking buffer [20 mM Tris, pH 7.5 (Fisher), 300 mM NaCl (Sigma), 0.1% (v/v) Tween 20 (Bio-Rad), 5% (w/v) skim milk powder, 0.05% (w/v) sodium azide (Fisher)], and then incubated for 1 h with rabbit polyclonal anti-gbCBG antibody at 1:3000 dilution in blocking buffer. Goat anti-rabbit IgG, conjugated to horseradish peroxidase (Bio-Rad), was used as the secondary antibody at 1:3000 dilution in blocking buffer, and protein band(s) were detected using ECL Plus western blotting detection reagent (GE Healthcare, Mississauga, ON, Canada). The protein bands were scanned using a Typhoon Variable Mode Imager (GE Healthcare) and were quantified by densitometry with AlphaEase 4.1 (Alpha Innotech Corp., San Leandro, CA, USA).
2.2.4. CBG determination using Western blotting For sex and metabolic state comparisons, 2.5 μg of albumin- and IgG-depleted serum from each sample was loaded per well to detect changes in CBG immunoreactivity using the protocol in Section 2.2.3. In samples where an electrophoretic doublet was observed, only the heavier band was quantified. CBG expression was calculated as % expression relative to a reference grizzly bear serum, which was also used to normalize for gel-to-gel variability.
2.3. Total serum cortisol and glucose assays Total serum cortisol concentrations were determined using a commercially available 125I cortisol radioimmunoassay (RIA) kit (MP Biomedicals, Irvine, CA, USA) validated for bear serum (Chow et al., 2010). The sensitivity of this kit is 2 ng cortisol/mL. Serum glucose concentrations in polar bears were determined using an Abbott Spectrum H Series II biochemistry analyzer (Abbott Laboratories Diagnostic Division).
2.4. Statistical analyses All descriptive statistics are presented as mean ± standard error of the means (SEM) and data were compared using either one-way analysis of variance (for comparison among sex and reproductive classes) or Student's t-test (for comparison between feeding and fasting states). Statistical significance was determined at p ≤ 0.05.
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3. Results The rabbit polyclonal anti-gbCBG antibody cross-reacted with polar bear CBG in a specific, concentration-dependent manner (Fig. 1). An electrophoretic doublet was observed in some samples (Figs. 2C and 3C). No significant differences were observed in serum total cortisol levels among sex and reproductive classes (F = 0.782, p = 0.47; n = 24; Fig. 2A). However, serum CBG expression was significantly elevated in lactating females relative to males and solitary females bears (F = 6.40, p ≤ 0.01; n = 24; Fig. 2B and C). The U/ C ratios of male polar bears captured while on sea ice or captured while on land were 46 ± 7 and 2.4 ± 0.42, respectively, and were significantly different (t = 7.04, p ≤ 0.001; n = 16). No significant differences were observed in serum total cortisol concentrations between bears in feeding and fasting states (t = 0.029, p = 0.98; n = 16; Fig. 3A). Serum CBG expression was almost two-fold higher in bears during fasting than during feeding (t = 3.29, p ≤ 0.01, n = 8; Fig. 3B and C). Further, serum glucose concentrations were lower in bears during fasting than during feeding (t = 2.044, p = 0.06; n = 15; Fig. 4). 4. Discussion We demonstrate for the first time that serum CBG expression is higher in fasting polar bears compared to those feeding. The crossreactivity between the anti-gbCBG antibody and polar bear sera was expected since grizzly and polar bears are closely related species and have diverged only recently (Talbot and Shields, 1996). The electrophoretic doublet that was observed in polar bears was also seen in grizzly bear sera (Chow et al., 2010). While the heavy band was always present, the lighter band was not consistently observed and was not included for CBG quantification. The origin of this doublet is unclear, but similar patterns have been observed in other animals and were due either to different isoforms or to post-translational modifications, including glycosylation (Berdusco et al., 1993; Mihrshahi et al., 2006). Serum total cortisol concentrations were similar across all of the sample groups and may be due to the acute stress of capture. This stress may lead to rapid elevations in serum cortisol concentration that may obscure baseline levels of this hormone (Romero and Reed, 2005), and the magnitude of this change may reflect the duration or magnitude of the stressor, as in the cortisol response to different types of capture in grizzly bears (Chow et al., 2010). All animals sampled in this study were captured by remote drug injection by darting from a helicopter, which may lead to our observations of similar serum total cortisol concentrations. However, we observed differences in CBG expression, which suggests that a serum concentration of this protein does not fluctuate as rapidly as cortisol with capture stress in polar bears and that we may be measuring the pre-capture baseline concentration. It has previously been shown that CBG expression may be altered hours after exposure to acute stressors in animals, including surgery and anesthesia in pigs (Dalin et al., 1993) and humans (Roth-Isigkeit et al., 2000), inescapable tail shocks in rats (Fleshner et al., 1995), and handling stress in Japanese quail (Malisch
Fig. 1. Western blot of serially diluted polar bear serum using anti-grizzly bear CBG antibody (1:3000 dilution). Lanes are: 1 — reference grizzly bear serum, 2.5 μg total serum protein), 2 — polar bear serum at 2.5 μg total serum protein loaded, 3 — polar bear serum at 5 μg total serum protein loaded, 4 — polar bear serum at 10 μg total serum protein loaded, 5–20 μg total serum protein loaded. An electrophoretic doublet can be observed for the polar bear serum, which suggests the existence of multiple isoforms of immunodetectable CBG in this species.
Fig. 2. Mean serum total cortisol concentration (A) and relative CBG expression (B) in adult female polar bears accompanied by dependent offspring (“Lactating”), solitary adult females (“Solitary”), and adult male (“Male”) polar bears showing individual data points (scatter plot) and the mean + SEM as a histogram (n = 24 with 8 per class). Bars with different letters are significantly different (p ≤ 0.05; one-way ANOVA). (C) Representative Western blot of Lactating, Solitary, and Male polar bears using anti-grizzly bear CBG antibody (1:3000 dilution). Total serum protein (2.5 μg) was loaded in each lane. A doublet is visible in most of the lanes, but only the higher molecular mass band was used for the quantification.
et al., 2010). Different capture methods have been shown to have no significant effect on CBG expression in grizzly bears (Chow et al., 2010). Thus, while total cortisol levels are affected by the stress of capture, our measurements of CBG expression in polar bears may reflect the true, pre-capture baseline expression of this protein, although further studies are required to confirm this finding. Sexual dimorphism in CBG expression has been reported previously in mammals (Fernandez-Real et al., 2002; Mataradze et al., 1992) and this is thought to be due to sex steroid modulation, including upregulation by estrogens and downregulation by androgens (Boonstra, 2005; White et al., 2006). The lack of a significant difference in expression between the non-lactating adult female and adult male bears in the present study may be related to their sampling time. For instance, these polar bears were captured in the southern Hudson Bay region during the ice-free season, suggesting that these animals were fasting. As fasting elevates CBG expression, this may have masked any sex-related changes in this protein's expression. Also, since these animals were captured after their mating season, the modulating effects of these steroids on CBG expression in polar bears may be unclear in this study. The basis for the elevated levels of CBG expression in the lactating female group relative to the other groups is unknown. We hypothesize that the additional metabolic demand associated with lactation while fasting may be involved in this response, but this remains to be tested.
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Fig. 3. Mean serum total cortisol concentration (A) and relative CBG expression (B) in male polar bears sampled while in feeding and fasting states showing individual data points (scatter plot) and the mean + SEM as a histogram (n = 16 with 8 per class; Student's t-test; p values are shown as inset). (C) Representative western blot of fasting and feeding polar bears using anti-grizzly bear CBG antibody (1:3000 dilution). 2.5 μg total serum protein was loaded in each lane. A doublet is clearly visible in the fasted groups, but only the higher molecular mass band was used for the quantification.
Serum total cortisol concentrations between feeding and fasting bears were not significantly different in this study, but the acute stress of capture may have masked baseline concentrations of this hormone. Studies in species that experience regular, seasonal fasts have reported lower serum total corticosteroid levels during fasting, including king penguins (Cherel et al., 1988a,b), migratory songbirds (Jenni et al., 2000), elephant (Champagne et al., 2006) and fur seals (Guinet et al., 2004). These decreases in corticosteroid levels may result in the suppression of catabolic processes and behaviours that
may be counterproductive to survival. In contrast, corticosteroid concentrations may be elevated in species where fasting is a stressor, including in red-legged kittiwake chicks (Kitaysky et al., 2001) and in humans (Beer et al., 1989). Additionally, CBG content has been found to decrease in animals in which fasting is a stressor, including rats (Tinnikov, 1999) and white-crowned sparrows (Lynn et al., 2003). However, total serum cortisol is elevated in the denning black bear (Harlow et al., 1990), a species that is considered to be highly adapted to seasonal food deprivation (Nelson, 1980). Our observation of elevated serum CBG expression in fasting polar bears suggests that a fasting adaptation may include a decrease in the bioavailability of corticosteroids either by reducing total corticosteroid levels and/or by elevating CBG content. This decrease in free corticosteroid (cortisol not bound to CBG) concentrations may lead to a downregulation of the catabolic pathways activated by this steroid hormone, resulting in an overall decrease in tissue metabolic capacity and energy substrate mobilization (Brillon et al., 1995). This adaptation may be particularly important to those subpopulations of polar bears in the seasonal ice and polar basin divergent ecoregions (Amstrup et al., 2007), where bears are forced ashore and undergo extended fasting annually as the sea ice melts completely or retreats from shore. In species that experience prolonged seasonal fasts, the associated decrease in energy expenditure may be an important adaptation to conserve endogenous energy stores, including proteins. Our results suggest that the elevation of serum CBG content and the associated reduction in cortisol bioavailability during fasting plays a key role in the protein sparing observed in polar bears (Ramsay et al., 1991; Atkinson and Ramsay, 1995; Lennox and Goodship, 2008). This finding is further supported by the lower serum glucose concentrations and a lack of an enhanced adrenocortical response in fasting state polar bears. Glucose is an important metabolic fuel and the concentration of this metabolite in circulation is elevated in response to chronic cortisol stimulation (Mommsen et al., 1999). This is due to the well-established role of cortisol in stimulating hepatic gluconeogenesis, including peripheral proteolysis and mobilization of amino acid stores as substrates for this glucose production (Mommsen et al., 1999; Sapolsky et al., 2000). Consequently, the decrease in serum glucose levels may reflect a lower hepatic gluconeogenic capacity, especially given that plasma cortisol levels were similar between the feeding and fasting animals. The reduction in free cortisol levels due to the higher CBG expression in the fasting animals may reduce target tissue response to cortisol stimulation, including downregulation of protein catabolism and amino acid mobilization. This adaptation would contribute to the sparing of protein during the extended fasting seen in polar bears (Ramsay et al., 1991; Atkinson and Ramsay, 1995; Lennox and Goodship, 2008; McCue, 2010). In conclusion, although total serum cortisol does not change with fasting relative to feeding animals, a fasting-associated increase in serum CBG expression may lead to a decrease in serum free cortisol levels. This change in cortisol dynamics may be a key metabolic adaptation to extended fasting in polar bears, leading to a reduction in target tissue response to cortisol stimulation, including peripheral proteolysis and liver gluconeogenesis. From a health monitoring stand point, measuring serum CBG levels in polar bears may provide important information on the nutritional status of the animal and/or its environmental conditions, especially given the recent finding that capture stress does not modulate the expression of this protein in grizzly bears (Chow et al., 2010). Acknowledgements
Fig. 4. Serum glucose concentration in polar bears sampled during feeding and fasting showing individual data points (scatter plot) and the mean + SEM as a histogram (n = 15 with 7–8 per group; Student's t test; p value is shown as inset).
For polar bear sera collected by the University of Saskatchewan, we thank S. Atkinson, S. Blum, and M. Ramsay. Funding for the field work in Ontario was provided by the Wildlife Research and Development Section of Ontario Ministry of Natural Resources (OMNR), Nunavut Department of Environment, Makivik Corporation, Safari Club International (Ontario
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Chapter), Safari Club International (Detroit Chapter), Les Brasseurs du Nord, La Fondation de la Faune du Québec, La Societé de la faune et des parcs du Québec, and OMNR's Climate Change Program: Projects CC-03/ 04-010, CC-04/05-002, and CC-05/06-036. We also acknowledge funding for this research provided by the National Science Foundation, USA, Natural Sciences and Engineering Research Council of Canada, Polar Continental Shelf Project, and World Wildlife Fund Canada. References Amstrup, S.C., Marcot, B.G., Douglas, D.C., 2007. Forecasting the range-wide status of polar bears at selected times in the 21st century. USGS Administrative Report U.S.G.S., Reston, Virginia. Atkinson, S.N., Ramsay, M.A., 1995. The effects of prolonged fasting of the body composition and reproductive success of female polar bears (Ursus maritimus). Funct. Ecol. 9, 559–567. Beer, S., Bircham, P., Bloom, S., Clark, P., Hales, C., Hughes, C., Jones, C., Marsh, D., Raggatt, P., Findlay, A., 1989. The effect of a 72-h fast on plasma levels of pituitary, adrenal, thyroid, pancreatic and gastrointestinal hormones in healthy men and women. J. Endocrinol. 120, 337–350. Berdusco, E.T.M., Hammond, G.L., Jacobs, R.A., Grolla, A., Akagi, K., Langlois, D., Challis, J.R.G., 1993. Glucocorticoid-induced increase in plasma corticosteroid-binding globulin levels in fetal sheep is associated with increased biosynthesis and alterations in glycosylation. Endocrinology 132, 2001–2008. Boonstra, R., 2005. Equipped for life: adaptive role of stress axis in male mammals. J. Mammal. 86, 236–247. Brillon, D., Zheng, B., Campbell, R., Matthews, D., 1995. Effect of cortisol on energy expenditure and amino acid metabolism in humans. Am. J. Physiol. Endocrinol. Metab. 268, E501–E513. Canadian Council on Animal Care, 2003. CCAC guidelines on: the care and use of wildlife. Canadian Council on Animal Care, Ottawa, Ontario, Canada. Cattet, M.R.L., Obbard, M.E., 2010. Use of hyaluronidase to improve chemical immobilization of free-ranging polar bears (Ursus maritimus). J. Wildl. Dis. 46, 246–250. Cattet, M.R.L., Caulkett, N.A., Polischuk, S.C., Ramsay, M.A., 1997. Reversible immobilization of free-ranging polar bears with medetomidine-zolazepam-tiletamine and atipamezole. J. Wildl. Dis. 33, 611–617. Champagne, C.D., Houser, D.S., Crocker, D.E., 2006. Glucose metabolism during lactation in a fasting animal, the northern elephant seal. Am. J. Physiol. Regul. Integr. Comp. Physiol. 291, 1129–1137. Cherel, Y., Leloup, J., Le Maho, Y., 1988a. Fasting in King Penguin. II. Hormonal and metabolic changes during molt. Am. J. Physiol. Regul. Integr. Comp. Physiol. 254, 178–184. Cherel, Y., Robin, J., Walch, O., Karmann, H., Netchitailo, P., Le Maho, Y., 1988b. Fasting in King Penguin. I. Hormonal and metabolic changes during breeding. Am. J. Physiol. Regul. Integr. Comp. Physiol. 254, 170–177. Chow, B.A., Hamilton, J., Alsop, D., Cattet, M.R.L., Stenhouse, G.B., Vijayan, M.M., 2010. Grizzly bear corticosteroid binding globulin: cloning and serum protein expression. Gen. Comp. Endocrinol. 167, 317–325. Dalin, A., Magnusson, U., Haggendal, J., Nyberg, L., 1993. The effect of thiopentone-sodium anesthesia and surgery, relocation, grouping, and hydrocortisone treatment on the blood levels of cortisol, corticosteroid-binding globulin, and catecholamines in pigs. J. Anim. Sci. 71, 1902–1909. Fernandez-Real, J.M., Pugeat, M., Grasa, M., Broch, M., Vendrell, J., Brun, J., Ricart, W., 2002. Serum corticosteroid-binding globulin concentration and insulin resistance syndrome: a population study. J. Clin. Endocrinol. Metab. 87, 4686–4690. Fleshner, M., Deak, T., Spencer, R.L., Laudenslager, M.L., Watkins, L.R., Maier, S.F., 1995. A long term increase in basal levels of corticosterone and a decrease in corticosteroidbinding globulin after acute stressor exposure. Endocrinology 136, 5336–5342. Gayrard, V., Alvinerie, M., Toutain, P.L., 1996. Interspecies variations of corticosteroidbinding globulin parameters. Domest. Anim. Endocrinol. 13, 35–45. Guinet, C., Servera, N., Mangin, S., Georges, J.Y., Lacroix, A., 2004. Change in plasma cortisol and metabolites during the attendance period ashore in fasting lactating subantarctic fur seals. Comp. Biochem. Physiol. A 137, 523–531. Harlow, H., Beck, T., Walters, L., Greenhouse, S., 1990. Seasonal serum glucose, progesterone, and cortisol levels of black bears (Ursus americanus). Can. J. Zool. 68, 183–187. Hellgren, E.C., 1998. Physiology of hibernation in bears. Ursus 10, 467–477.
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Comparative Biochemistry and Physiology, Part A j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a
Serum corticosteroid binding globulin expression is modulated by fasting in polar bears (Ursus maritimus) Brian A. Chow a, Jason Hamilton a, Marc R.L. Cattet b, Gordon Stenhouse c, Martyn E. Obbard d, Mathilakath M. Vijayan a,⁎ a
Department of Biology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada Canadian Cooperative Wildlife Health Centre, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5B4, Canada Foothills Research Institute, Hinton, Alberta, T7V 1X7, Canada d Wildlife Research and Development Section, Ontario Ministry of Natural Resources, Trent University, Peterborough, Ontario, K9J 7B8, Canada b c
a r t i c l e
i n f o
Article history: Received 11 May 2010 Received in revised form 16 September 2010 Accepted 20 September 2010 Available online 29 September 2010 Keywords: CBG Cortisol Fasting Protein expression Stress response Ursids
a b s t r a c t Polar bears (Ursus maritimus) from several subpopulations undergo extended fasting during the ice-free season. However, the animals appear to conserve protein despite the prolonged fasting, though the mechanisms involved are poorly understood. We hypothesized that elevated concentrations of corticosteroid binding globulin (CBG), the primary cortisol binding protein in circulation, lead to cortisol resistance and provide a mechanism for protein conservation during extended fasting. The metabolic state (feeding vs. fasting) of 16 field sampled male polar bears was determined based on their serum urea to creatinine ratio (N 25 for feeding vs. b 5 for fasting). There were no significant differences in serum cortisol levels between all male and female polar bears sampled. Serum CBG expression was greater in lactating females relative to nonlactating females and males. CBG expression was significantly higher in fasting males when compared to nonfasting males. This leads us to suggest that CBG expression may serve as a mechanism to conserve protein during extended fasting in polar bears by reducing systemic free cortisol concentrations. This was further supported by a lower serum glucose concentration in the fasting bears. As well, a lack of an enhanced adrenocortical response to acute capture stress supports our hypothesis that chronic hunger is not a stressor in this species. Overall, our results suggest that elevated serum CBG expression may be an important adaptation to spare proteins by limiting cortisol bioavailability during extended fasting in polar bears. © 2010 Elsevier Inc. All rights reserved.
1. Introduction The polar bear (Ursus maritimus) is a large arctic carnivore that occupies the top trophic level in a marine food web (Hobson and Welch, 1992). In the Canadian arctic, they primarily feed on seals (Phocidae) and rely on sea ice as a seasonal platform upon which they hunt their prey. During the ice-free period (Jun–Oct), populations are forced ashore or onto unproductive multi-year ice, where bears generally undergo extended fasting (Stirling and Derocher, 1993). Although polar bears have been observed to forage while on land, it is generally accepted that these supplemental food sources do not contribute significantly to their diet (Lunn and Stirling, 1985). As year-round opportunistic feeders, polar bears are known to enter a facultative hypometabolic state that is similar to hibernation in American black (U. americanus) and grizzly bears (U. arctos) during periods of sparse food availability, as evidenced by the lowering of heart rate, body temperature, and serum urea to creatinine (U/C) ratio
⁎ Corresponding author. Tel.: + 1 519 888 4567x32035; fax: + 1 519 746 0614. E-mail address: [email protected] (M.M. Vijayan). 1095-6433/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2010.09.017
(Hellgren, 1998; Ramsay et al., 1991). However, unlike these species, most polar bears may remain in an active, “walking hibernation” state while fasting. An exception is pregnant females, which will enter maternity dens to give birth and initiate lactation during this period (Nelson et al., 1983). Corticosteroids are thought to play an important role during extended fasting in animals by mobilizing energy substrates, including amino acids from proteins to fuel metabolic processes critical for survival (Schwartz et al., 1995). Corticosteroids are secreted from the adrenal cortex in response to stressor-induced activation of the hypothalamus–pituitary–adrenal (HPA) axis. The primary step involves the release of corticotropin releasing hormone from the hypothalamus in response to sensory neural inputs, stimulating the pituitary gland to release adrenocorticotrophic hormone. This peptide then activates melanocortin 2 receptors on the adrenal cortex, leading to cortisol biosynthesis (Sapolsky et al., 2000). Elevation in serum corticosteroid levels stimulates muscle protein catabolism and enhances gluconeogenesis especially from amino acids in the liver, resulting in hyperglycemia (Brillon et al., 1995; Mommsen et al., 1999). However, little is known about the role of cortisol in the metabolic adaptation of bears to extended fasting.
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It is known that the levels of serum total cortisol, the principal corticosteroid in bears, are elevated in denning black bears relative to summer active bears (Harlow et al., 1990). However, despite hypercortisolemia, black bears conserve their lean (protein) body mass during denning (Hellgren, 1998; Lohuis et al., 2007), leading us to hypothesize that these animals may be resistant to corticosteroid action during extended fasting. Studies have shown that in mammals up to 95% of circulating corticosteroid is bound to a 55 kDa glycoprotein, corticosteroid-binding globulin (CBG) (Gayrard et al., 1996; Rosner, 1990). The binding of the steroid to CBG reduces the free cortisol concentration, which is the biologically active fraction. Consequently, serum CBG concentrations play a major role in regulating corticosteroid bioavailability and target tissue responses (Rosner, 1990). Against this background, we tested the hypothesis that serum CBG expression is higher in fasting polar bears as an adaptation to limit cortisol bioavailability and protein catabolism. In addition, we compared serum CBG expression in adult male and female polar bears to explore differences associated with sexual dimorphism and confirm previous findings in other mammals (Mataradze et al., 1992; Fernandez-Real et al., 2002; Chow et al., 2010). 2. Materials and methods 2.1. Polar bear serum samples We obtained archived frozen serum samples (2–4 mL per bear) from 40 polar bears (n = 24 males, 16 females) that had been captured and released in conjunction with long-term research projects in the Canadian Arctic conducted by the University of Saskatchewan from 1989 to 1997, and the Ontario Ministry of Natural Resources from 2002 to 2009. All bears were located and immobilized from a helicopter by remote drug delivery (Palmer Cap-Chur Inc., Powder Springs, GA, USA) as detailed in Cattet et al. (1997) and Cattet and Obbard (2010). Samples were chilled on icepacks in a cooler immediately following blood collection to slow down blood cell metabolism. All samples were collected and processed similarly, with sera frozen (−25 °C) within 8 h of collection in long-term storage at the University of Saskatchewan. The research protocols for both projects were approved annually by animal care committees at the University of Saskatchewan and Ontario Ministry of Natural Resources and was in accordance with guidelines provided by the Canadian Council on Animal Care (2003) for the safe handling of wildlife. 2.2. Serum CBG expression 2.2.1. Comparison by sex To evaluate sexual dimorphism in CBG expression in polar bears, we compared levels in samples collected from 24 polar bears captured from the area of southern Hudson Bay during the months of September and October. All samples were from adult (≥5 years) bears of similar body condition within three sex and reproductive classes: lactating females accompanied by dependent offspring (“lactating females”, n = 8), non-lactating solitary females (“solitary females”, n = 8), and males (n = 8). 2.2.2. Comparison by metabolic state To compare CBG levels between feeding and fasting states, samples were collected from 16 adult (≥5 years) male polar bears. Eight animals were captured on sea ice during April and May, a time when most bears are in a feeding state, from the western Hudson Bay (WHB) and Lancaster Sound (LS) subpopulations. Another eight animals were captured on land from the WHB subpopulation during October, when most polar bears are in a fasting state. Serum urea to creatinine (U/C) ratios have been shown to be a reliable marker of metabolic state in polar bears (Ramsay et al., 1991). We determined serum urea and creatinine concentrations using an Abbott Spectrum H
Series II biochemistry analyzer (Abbott Laboratories Diagnostic Division, Abbott Park, IL, USA) to confirm the metabolic state of bears at the time of capture.
2.2.3. Validation of CBG cross-reactivity in polar bears A peptide region of a previously sequenced grizzly bear CBG was used to generate an affinity purified rabbit polyclonal antibody, which was then used to test for cross-reactivity in polar bears as previously described (Chow et al., 2010). Specifically, polar bear serum was depleted of albumin and IgG with Aurum Serum Protein Mini-kits (Bio-Rad, Hercules, CA, USA) and total protein concentration was determined by the bicinchoninic acid method (Smith et al., 1985) using bovine serum albumin as the standard. Samples were serially diluted to 2.5, 5, 10, and 20 μg total serum protein in sample buffer [0.06 M Tris-HCl (pH 6.8), 25% (v/v) glycerol, 0.02% (w/v) SDS, 0.001% (w/v) bromophenol blue (Fisher Scientific, Fair Lawn, NJ, USA)] and loaded into wells on 10% reducing polyacrylamide gels for immunodetection. A low-range molecular weight marker (Bio-Rad) was used to confirm molecular mass of the protein. Proteins were separated (200 V for 50 min; Mini Protean III, Bio-Rad) using a discontinuous buffer (Laemmli, 1970). The separated proteins were transferred to a 0.22 μm pore size nitrocellulose membrane using a Transblot SD SemiDry Electrophoretic Transfer Cell (Bio-Rad). Equal loading and transfer efficiency were inspected by Ponceau S (Bio-Rad) staining of the membrane. The membranes were rinsed and blocked with blocking buffer [20 mM Tris, pH 7.5 (Fisher), 300 mM NaCl (Sigma), 0.1% (v/v) Tween 20 (Bio-Rad), 5% (w/v) skim milk powder, 0.05% (w/v) sodium azide (Fisher)], and then incubated for 1 h with rabbit polyclonal anti-gbCBG antibody at 1:3000 dilution in blocking buffer. Goat anti-rabbit IgG, conjugated to horseradish peroxidase (Bio-Rad), was used as the secondary antibody at 1:3000 dilution in blocking buffer, and protein band(s) were detected using ECL Plus western blotting detection reagent (GE Healthcare, Mississauga, ON, Canada). The protein bands were scanned using a Typhoon Variable Mode Imager (GE Healthcare) and were quantified by densitometry with AlphaEase 4.1 (Alpha Innotech Corp., San Leandro, CA, USA).
2.2.4. CBG determination using Western blotting For sex and metabolic state comparisons, 2.5 μg of albumin- and IgG-depleted serum from each sample was loaded per well to detect changes in CBG immunoreactivity using the protocol in Section 2.2.3. In samples where an electrophoretic doublet was observed, only the heavier band was quantified. CBG expression was calculated as % expression relative to a reference grizzly bear serum, which was also used to normalize for gel-to-gel variability.
2.3. Total serum cortisol and glucose assays Total serum cortisol concentrations were determined using a commercially available 125I cortisol radioimmunoassay (RIA) kit (MP Biomedicals, Irvine, CA, USA) validated for bear serum (Chow et al., 2010). The sensitivity of this kit is 2 ng cortisol/mL. Serum glucose concentrations in polar bears were determined using an Abbott Spectrum H Series II biochemistry analyzer (Abbott Laboratories Diagnostic Division).
2.4. Statistical analyses All descriptive statistics are presented as mean ± standard error of the means (SEM) and data were compared using either one-way analysis of variance (for comparison among sex and reproductive classes) or Student's t-test (for comparison between feeding and fasting states). Statistical significance was determined at p ≤ 0.05.
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3. Results The rabbit polyclonal anti-gbCBG antibody cross-reacted with polar bear CBG in a specific, concentration-dependent manner (Fig. 1). An electrophoretic doublet was observed in some samples (Figs. 2C and 3C). No significant differences were observed in serum total cortisol levels among sex and reproductive classes (F = 0.782, p = 0.47; n = 24; Fig. 2A). However, serum CBG expression was significantly elevated in lactating females relative to males and solitary females bears (F = 6.40, p ≤ 0.01; n = 24; Fig. 2B and C). The U/ C ratios of male polar bears captured while on sea ice or captured while on land were 46 ± 7 and 2.4 ± 0.42, respectively, and were significantly different (t = 7.04, p ≤ 0.001; n = 16). No significant differences were observed in serum total cortisol concentrations between bears in feeding and fasting states (t = 0.029, p = 0.98; n = 16; Fig. 3A). Serum CBG expression was almost two-fold higher in bears during fasting than during feeding (t = 3.29, p ≤ 0.01, n = 8; Fig. 3B and C). Further, serum glucose concentrations were lower in bears during fasting than during feeding (t = 2.044, p = 0.06; n = 15; Fig. 4). 4. Discussion We demonstrate for the first time that serum CBG expression is higher in fasting polar bears compared to those feeding. The crossreactivity between the anti-gbCBG antibody and polar bear sera was expected since grizzly and polar bears are closely related species and have diverged only recently (Talbot and Shields, 1996). The electrophoretic doublet that was observed in polar bears was also seen in grizzly bear sera (Chow et al., 2010). While the heavy band was always present, the lighter band was not consistently observed and was not included for CBG quantification. The origin of this doublet is unclear, but similar patterns have been observed in other animals and were due either to different isoforms or to post-translational modifications, including glycosylation (Berdusco et al., 1993; Mihrshahi et al., 2006). Serum total cortisol concentrations were similar across all of the sample groups and may be due to the acute stress of capture. This stress may lead to rapid elevations in serum cortisol concentration that may obscure baseline levels of this hormone (Romero and Reed, 2005), and the magnitude of this change may reflect the duration or magnitude of the stressor, as in the cortisol response to different types of capture in grizzly bears (Chow et al., 2010). All animals sampled in this study were captured by remote drug injection by darting from a helicopter, which may lead to our observations of similar serum total cortisol concentrations. However, we observed differences in CBG expression, which suggests that a serum concentration of this protein does not fluctuate as rapidly as cortisol with capture stress in polar bears and that we may be measuring the pre-capture baseline concentration. It has previously been shown that CBG expression may be altered hours after exposure to acute stressors in animals, including surgery and anesthesia in pigs (Dalin et al., 1993) and humans (Roth-Isigkeit et al., 2000), inescapable tail shocks in rats (Fleshner et al., 1995), and handling stress in Japanese quail (Malisch
Fig. 1. Western blot of serially diluted polar bear serum using anti-grizzly bear CBG antibody (1:3000 dilution). Lanes are: 1 — reference grizzly bear serum, 2.5 μg total serum protein), 2 — polar bear serum at 2.5 μg total serum protein loaded, 3 — polar bear serum at 5 μg total serum protein loaded, 4 — polar bear serum at 10 μg total serum protein loaded, 5–20 μg total serum protein loaded. An electrophoretic doublet can be observed for the polar bear serum, which suggests the existence of multiple isoforms of immunodetectable CBG in this species.
Fig. 2. Mean serum total cortisol concentration (A) and relative CBG expression (B) in adult female polar bears accompanied by dependent offspring (“Lactating”), solitary adult females (“Solitary”), and adult male (“Male”) polar bears showing individual data points (scatter plot) and the mean + SEM as a histogram (n = 24 with 8 per class). Bars with different letters are significantly different (p ≤ 0.05; one-way ANOVA). (C) Representative Western blot of Lactating, Solitary, and Male polar bears using anti-grizzly bear CBG antibody (1:3000 dilution). Total serum protein (2.5 μg) was loaded in each lane. A doublet is visible in most of the lanes, but only the higher molecular mass band was used for the quantification.
et al., 2010). Different capture methods have been shown to have no significant effect on CBG expression in grizzly bears (Chow et al., 2010). Thus, while total cortisol levels are affected by the stress of capture, our measurements of CBG expression in polar bears may reflect the true, pre-capture baseline expression of this protein, although further studies are required to confirm this finding. Sexual dimorphism in CBG expression has been reported previously in mammals (Fernandez-Real et al., 2002; Mataradze et al., 1992) and this is thought to be due to sex steroid modulation, including upregulation by estrogens and downregulation by androgens (Boonstra, 2005; White et al., 2006). The lack of a significant difference in expression between the non-lactating adult female and adult male bears in the present study may be related to their sampling time. For instance, these polar bears were captured in the southern Hudson Bay region during the ice-free season, suggesting that these animals were fasting. As fasting elevates CBG expression, this may have masked any sex-related changes in this protein's expression. Also, since these animals were captured after their mating season, the modulating effects of these steroids on CBG expression in polar bears may be unclear in this study. The basis for the elevated levels of CBG expression in the lactating female group relative to the other groups is unknown. We hypothesize that the additional metabolic demand associated with lactation while fasting may be involved in this response, but this remains to be tested.
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Fig. 3. Mean serum total cortisol concentration (A) and relative CBG expression (B) in male polar bears sampled while in feeding and fasting states showing individual data points (scatter plot) and the mean + SEM as a histogram (n = 16 with 8 per class; Student's t-test; p values are shown as inset). (C) Representative western blot of fasting and feeding polar bears using anti-grizzly bear CBG antibody (1:3000 dilution). 2.5 μg total serum protein was loaded in each lane. A doublet is clearly visible in the fasted groups, but only the higher molecular mass band was used for the quantification.
Serum total cortisol concentrations between feeding and fasting bears were not significantly different in this study, but the acute stress of capture may have masked baseline concentrations of this hormone. Studies in species that experience regular, seasonal fasts have reported lower serum total corticosteroid levels during fasting, including king penguins (Cherel et al., 1988a,b), migratory songbirds (Jenni et al., 2000), elephant (Champagne et al., 2006) and fur seals (Guinet et al., 2004). These decreases in corticosteroid levels may result in the suppression of catabolic processes and behaviours that
may be counterproductive to survival. In contrast, corticosteroid concentrations may be elevated in species where fasting is a stressor, including in red-legged kittiwake chicks (Kitaysky et al., 2001) and in humans (Beer et al., 1989). Additionally, CBG content has been found to decrease in animals in which fasting is a stressor, including rats (Tinnikov, 1999) and white-crowned sparrows (Lynn et al., 2003). However, total serum cortisol is elevated in the denning black bear (Harlow et al., 1990), a species that is considered to be highly adapted to seasonal food deprivation (Nelson, 1980). Our observation of elevated serum CBG expression in fasting polar bears suggests that a fasting adaptation may include a decrease in the bioavailability of corticosteroids either by reducing total corticosteroid levels and/or by elevating CBG content. This decrease in free corticosteroid (cortisol not bound to CBG) concentrations may lead to a downregulation of the catabolic pathways activated by this steroid hormone, resulting in an overall decrease in tissue metabolic capacity and energy substrate mobilization (Brillon et al., 1995). This adaptation may be particularly important to those subpopulations of polar bears in the seasonal ice and polar basin divergent ecoregions (Amstrup et al., 2007), where bears are forced ashore and undergo extended fasting annually as the sea ice melts completely or retreats from shore. In species that experience prolonged seasonal fasts, the associated decrease in energy expenditure may be an important adaptation to conserve endogenous energy stores, including proteins. Our results suggest that the elevation of serum CBG content and the associated reduction in cortisol bioavailability during fasting plays a key role in the protein sparing observed in polar bears (Ramsay et al., 1991; Atkinson and Ramsay, 1995; Lennox and Goodship, 2008). This finding is further supported by the lower serum glucose concentrations and a lack of an enhanced adrenocortical response in fasting state polar bears. Glucose is an important metabolic fuel and the concentration of this metabolite in circulation is elevated in response to chronic cortisol stimulation (Mommsen et al., 1999). This is due to the well-established role of cortisol in stimulating hepatic gluconeogenesis, including peripheral proteolysis and mobilization of amino acid stores as substrates for this glucose production (Mommsen et al., 1999; Sapolsky et al., 2000). Consequently, the decrease in serum glucose levels may reflect a lower hepatic gluconeogenic capacity, especially given that plasma cortisol levels were similar between the feeding and fasting animals. The reduction in free cortisol levels due to the higher CBG expression in the fasting animals may reduce target tissue response to cortisol stimulation, including downregulation of protein catabolism and amino acid mobilization. This adaptation would contribute to the sparing of protein during the extended fasting seen in polar bears (Ramsay et al., 1991; Atkinson and Ramsay, 1995; Lennox and Goodship, 2008; McCue, 2010). In conclusion, although total serum cortisol does not change with fasting relative to feeding animals, a fasting-associated increase in serum CBG expression may lead to a decrease in serum free cortisol levels. This change in cortisol dynamics may be a key metabolic adaptation to extended fasting in polar bears, leading to a reduction in target tissue response to cortisol stimulation, including peripheral proteolysis and liver gluconeogenesis. From a health monitoring stand point, measuring serum CBG levels in polar bears may provide important information on the nutritional status of the animal and/or its environmental conditions, especially given the recent finding that capture stress does not modulate the expression of this protein in grizzly bears (Chow et al., 2010). Acknowledgements
Fig. 4. Serum glucose concentration in polar bears sampled during feeding and fasting showing individual data points (scatter plot) and the mean + SEM as a histogram (n = 15 with 7–8 per group; Student's t test; p value is shown as inset).
For polar bear sera collected by the University of Saskatchewan, we thank S. Atkinson, S. Blum, and M. Ramsay. Funding for the field work in Ontario was provided by the Wildlife Research and Development Section of Ontario Ministry of Natural Resources (OMNR), Nunavut Department of Environment, Makivik Corporation, Safari Club International (Ontario
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