Oecologia https://doi.org/10.1007/s00442-020-04645-5 PHYSIOLOGICAL ECOLOGY – ORIGINAL RESEARCH Fasting afects amino acid nitrogen isotope values: a new tool for identifying nitrogen balance of free‑ranging mammals Nico Lübcker1 · John P. Whiteman2,4 · Robert P. Millar3 · P. J. Nico de Bruyn1 · Seth D. Newsome4 Received: 5 August 2019 / Accepted: 7 April 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020 Abstract Changes in the nutritional status of free-ranging animals have a strong infuence on individual ftness, yet it remains challeng- ing to monitor longitudinally. Nitrogen (δ15N) and carbon (δ13C) isotope values measured chronologically along the length of metabolically inert keratinous tissues can be used as a nutritional biomarker to retrospectively reconstruct the foraging ecology and eco-physiology of consumers. We quantitatively describe the physiological efects of fasting on amino acid metabolism using sequentially measured bulk tissue and amino acid δ15N values along the length of whiskers sampled from free-ranging juvenile, subadults, adult female, and male southern elephant seals (SES; Mirounga leonina) on Marion Island in the Southern Ocean. For both juveniles and adult females, whisker segments representing fasting had signifcantly higher bulk tissue δ15N values of 0.6 ± 0.5‰ and 1.3–1.8‰, respectively, in comparison to segments unafected by fasting. We also found a large increase (2–6‰) in δ15N values for most glucogenic amino acids and a simultaneous depletion (2–3‰) of alanine in segments refecting fasting, which enabled us to accurately predict (74%) the nutritional status of our model species. We hypothesize that the glucose-alanine cycle is the mechanism driving the observed depletion of alanine δ15N val- ues during fasting. We demonstrated that keratinaceous tissues can be used as a longitudinal nutritional biomarker to detect changes in the nitrogen balance of an individual. Moreover, it is evident that physiological factors have an important infuence on tissue δ15N values and can lead to erroneous bulk tissue or amino acid isotope-based reconstructions of foraging habits. Keywords Amino acid metabolism · Compound-specifc stable isotopes · Diet · Elephant seals · Nutritional biomarker Introduction Animals often experience prolonged fasts during which met- abolic processes regulate changes in the utilization of stored Communicated by Helene Marsh. endogenous substrates such as skeletal muscle (proteins) and adipose tissue (lipids) (Raubenheimer et al. 2009; Fontana Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s0044 2-020-04645 -5) contains and Partridge 2015; Secor and Carey 2011; Vidal et al. supplementary material, which is available to authorized users. 2019). Ultimately, the integrative physiological response to extended fasting bouts can have strong impacts on individual * Nico Lübcker ftness, and are thus intrinsically linked to their evolution [email protected] (Secor and Carey 2011). Yet, assessing the relative impor- 1 Department of Zoology and Entomology, Mammal tance of various biochemical and physiological mechanisms Research Institute, University of Pretoria, Private Bag X20, that enable organisms to maintain physiological homeostasis Hatfeld 0028, South Africa while solely relying on endogenous resources remains a con- 2 Department of Biological Sciences, Old Dominion tentious topic (Secor and Carey 2011; Lee et al. 2012). Fur- University, Norfolk, VA 23529, USA thermore, our understanding of such mechanisms that enable 3 Centre for Neuroendocrinology and Department free-ranging animals to withstand and recover from extended of Immunology, Faculty of Health Sciences, University fasts are often hampered by the challenge of building longi- of Pretoria, Pretoria 0001, South Africa tudinal (temporal) records of their nutritional status. 4 Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA Vol.:(0123456789)1 3 Oecologia Biomarkers can refect changes in how animals catabolize catabolism during fasting and thus will show minimal to no or synthesize molecules during fasting (Petzke et al. 2010), change in δ15N because they are not closely associated with and chemical analysis of such compounds can provide valu- nitrogen cycling and cannot easily be deaminated without able insights on the relationship between an organism’s phys- catabolizing the entire compound. In summary, we predict iology, diet, and environmental conditions (Fuller et al. 2005; that the ofsets between the δ15N of trophic and source amino Mekota et al. 2009; Hatch 2012; Ohkouchi et al. 2017). For acids in fasting animals are expected to be larger than in ani- example, nitrogen (δ15N) and carbon (δ13C) isotope analysis mals that are consuming exogenous resources. of tissues provide information about resource and habitat use, Adult southern elephant seals (SES; Mirounga leonina) as well as physiological status (Jaeger et al. 2010; Newsome experience two distinct prolonged fasting periods during their et al. 2010; O’Brien 2015). While δ13C values are often used annual life cycle (Fig. 1S; Le Boeuf and Laws 1994). One to infer sources of primary production that fuel the food webs occurs during the breeding season (Oct.–Nov.) which ends that animals rely on, δ15N values are commonly used to esti- with a brief foraging bout, and the other during the annual mate trophic level (Peterson and Fry 1987). The pattern of obligatory molt (Dec.–Jan.). Tissues synthesized while fast- trophic enrichment in tissue δ15N is a result of fractionation ing provide a means to examine the efects of protein catabo- 15 among the amino radicals (–NH2) that are produced during lism on nitrogen balance and by extension on δ N values amino acid catabolism; the isotopically depleted nitrogen of the consumer. After birth, juvenile SES pups suckle for (14N) is preferentially excreted in the form of ammonia, urea, 22–23 days before undergoing a post-weaning fast that or uric acid (Peterson and Fry 1987) and the retained 15N is lasts ~ 40–60 days (Wilkinson and Bester 1990). During this then incorporated into body nitrogen pools and used to build fast, recently-weaned pups lose up to ~ 30% of their original and maintain tissues. Ofsets in δ15N values between the ani- weaning body mass (Wilkinson and Bester 1990; Carlini mals and their food are referred to as trophic discrimination et al. 2001) before departing on their frst foraging trip; the factors (TDF; Peterson and Fry 1987), which vary depending mass lost consists of 39% water, 47% fat, and 12% protein. on the type of nitrogenous waste compound produced (Van- Using estimates of whisker growth rates (Lubcker et al. 2016), derklift and Ponsard 2003), as well as the quantity and quality we showed that δ15N values of longitudinally sub-sampled of protein in their diets (Hughes et al. 2018). whisker segments from recently-weaned SES pups showed In addition to diet composition, physiological processes enrichment of ~ 1.0‰ while fasting (Lübcker et al. 2017). such as starvation or gestation can also afect the isotopic Here we used a combined bulk tissue and compound- composition of tissues (Hobson et al. 1993; Fuller et al. 2004, specifc isotope approach to advance our understanding of 2005). Recent meta-analyses demonstrated that fasting causes physiological changes in amino acid metabolism that occur a small yet measurable increase in bulk tissue mean (± SD) during fasting, and potentially establish amino acid δ15N δ15N values of + 0.5 ± 0.2‰ (McCue 2010; Hertz et al. 2015) analysis as a robust proxy for identifying the use of endoge- as the individual essentially ‘consumes’ itself by cataboliz- nous versus exogenous resources. We frst compared how the ing its protein- (skeletal muscle) and lipid-rich (adipose) tis- δ15N of amino acids changed during known catabolic and sues to fuel metabolism (Fuller et al. 2005). Theoretically, anabolic states that were identifed using bulk tissue δ15N isotopic analysis of individual compounds may provide data measured chronologically along the length of juvenile additional information about the biochemical mechanisms SES whiskers with known whisker growth histories. We then that enable animals to fast for prolonged periods (Ohkouchi used these data to determine if fasting infuenced the δ15N et al. 2017; Whiteman et al. 2019). For animals that do not values measured along the length of whiskers sampled from consume appreciable amounts of glucose (e.g., carnivores), free-ranging adult SES with unknown whisker growth his- amino acids are catabolized for glucose synthesis via gluco- tories. Although we have previously described the growth neogenesis to fuel activity, which results in the preferential rate and shedding phenology of SES whiskers (Lübcker et al. deamination and excretion of 14N and yields the measurable 2016), it is still challenging to ascertain when the whisker TDFs described above. During fasting, endogenous protein growth of free-ranging seals commences (McHuron et al. (i.e., skeletal muscle), which is 15N-enriched relative to 2019). Identifying the segment(s) of the whisker grown exogenous dietary protein, becomes the primary substrate while fasting on land could clarify the whisker growth his- for gluconeogenesis (Krebs 1964) and consequently enriches tories and refne the use of this tissue to provide longitudinal the δ15N of the remaining amino acids in the body pool that physiological and ecological information (McHuron et al. are used to synthesize tissues.