Thyroxine levels in agouti and non-agouti deer mice ž / Peromyscus maniculatus

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1 Ž. Comparative Biochemistry and Physiology Part A Thyroxine levels in agouti and non-agouti deer mice ž / Peromyscus maniculatus Joy M. Lapseritis a, Virginia Hayssen b, a Department of Biology, Smith College, Northampton, MA 01063, USA and Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA b Department of Biology, Smith College, Northampton, MA 01063, USA Received 9 February 2001; received in revised form 7 June 2001; accepted 11 June 2001 Abstract Total thyroxine was assessed by radioimmunoassay for 58 female deer mice of two subspecies ŽPeromyscus maniculatus bairdii and P. m. gracilis. and two color morphs Ž agouti and non-agouti.. P. m. bairdii of both color-morphs had significantly higher mean thyroxine levels than P. m. gracilis. Non-agouti deer mice of both subspecies had significantly higher mean thyroxine levels than agouti deer mice. This is the first report of thyroid hormone differences associated with the non-agouti allele Elsevier Science Inc. All rights reserved. Keywords: Thyroxine; Peromyscus; Obesity; Agouti; Non-agouti; Pleiotropy; Coat-color; Melanocortin; Melanocyte-stimulating Hormone; Extension-locus 1. Introduction The agouti gene affects coat-color in nearly all mammals Ž Searle, 1968; Silvers, Wild-type Ž agouti. pelage is characterized by hairs with an apical or sub-apical yellow band on otherwise black hairs. Agouti protein functions locally at the hair follicle as a paracrine hormone that antagonizes the action of melanocyte-stimulating hormone Ž MSH. at the melanocortin-1 receptor, itself the product of the extension locus ŽOllmann et al., Mammals homozygous for the recessive extreme non-agouti allele Ž non-agouti. do not ex- Corresponding author. Tel.: ; fax: address: vhayssen@science.smith.edu Ž V. Hayssen.. press agouti protein due to a mutation at the start codon Ž Mus, Hustad et al., 1995., and lack yellow bands on individual hairs ŽSearle, 1968; Horner et al., In effect, these non-agouti animals are a natural knock-out for the agouti gene. In addition to pelage differences, non-agouti deer mice Ž Peromyscus maniculatus. differ in activity levels, brain catecholamines, spleen size, and body mass in comparison with wild-type, agouti animals ŽHayssen, 1992, 1994, 1997, 1998; Hayssen et al., Non-agouti female deer mice ŽP. m. gracilis. are especially large, with many over 40 g in weight Ž Hayssen, 1996a,b.. Ž vy Dominant mutations e.g. A. at the agouti locus in laboratory mice Ž Mus musculus. are associated with obesity. Over-expression of agouti protein leads to hyperphagia, inhibited lipolysis, and increased triglyceride storage in adipocytes $ - see front matter 2001 Elsevier Science Inc. All rights reserved. Ž. PII: S

2 296 J.M. Lapseritis, V. Hayssen Comparati e Biochemistry and Physiology Part A Ž Klebig et al., 1995; Xue et al., Presumably, the agouti protein antagonizes MSH at melanocortin receptors in neural and other tissues in agouti animals to mediate these effects. The same explanation cannot explain the propensity for non-agouti deermice to become heavy, as these animals do not express the agouti protein; furthermore, increased body mass is observed only in females Žmales of either color morph rarely become obese.. The amino acid tyrosine is the precursor of thyroxine, and agouti protein is an antagonist at several levels in the tyrosine metabolism cascade. Agouti protein may be responsible for differences in brain catecholamines in agouti and non-agouti deermice Ž Hayssen et al., Glycogenic and ketogenic catabolites of tyrosine contribute to fatty acid synthesis pathways, consistent with the results of over-expression of agouti ŽKlebig et al., 1995; Xue et al., 1998; Claycombe et al., 2000a.. Given the pervasiveness of agouti activity in tyrosine metabolism, a role for agouti in thyroxine anabolism is logical. Thyroxine is a major metabolic regulator, and interactions with agouti may be responsible for body mass differences. The presumed absence of agouti protein in non-agouti deermice reduces the likelihood of a fat-metabolism mechanism in weight gain as seen in A vy mice, implicating thyroid hypo-activity. In this study, we measured thyroid hormone of female agouti and non-agouti deermice of two subspecies Ž Peromyscus maniculatus gracilis and P. m. bairdii. to better understand the pleiotropic effects of the agouti locus and the metabolic endocrinology of body mass in non-agouti deermice. 2. Materials and methods 2.1. Source, husbandry, and physical characteristics of animals The non-agouti allele was discovered in Peromyscus maniculatus in the subspecies gracilis from the Horner Colony at Smith College, Northampton, MA Ž Horner et al., The allele was moved to the P. m. bairdii genetic background at the Peromyscus Genetic Stock Center ŽPGSC, University of South Carolina, Columbia, SC.. Thus, the non-agouti genotype is available in two subspecific backgrounds Žbairdii and gracilis.. Husbandry practices and colony maintenance differ at the two facilities Žsee Hayssen, 1998 for P. m. gracilis; and Joyner et al., 1998 for P. m. bairdii.. Even distributions of sample size, age, and body mass were not possible because the different subspecies were from different colonies in different states with different culling regimes. Horner colony deermice Ž P. m. gracilis. are maintained with longer lifespans than at PGSC Ž P. m. bairdii.. The bairdii animals are culled when they are young; the gracilis animals are allowed to live until they effectively die of old age at 4 6 years. Thus, the age distributions in the two colonies are very different. Age-matching is not possible because no old bairdii were alive to sample. Similarly, obesity could not be matched between the two subspecies because heavy animals are not present in the bairdii colony, but are of primary interest in the gracilis colony. Finally, more gracilis were sampled because the authors maintain this colony and were able to collect more samples. Also, we were primarily interested in agouti and non-agouti gracilis because we have a substantial body of data on pleiotropic effects of the agouti locus in this genetic background. Thus, the P. m. gracilis sample was much older and slightly heavier than that of P. m. bairdii. P. m. gracilis individuals ranged in age from 115 to 1622 days Ž mean 865 days., and P. m. bairdii from 86 to 361 days Ž mean 151 days.. Total body mass in P. m. gracilis ranged from 11.6 to 40 g Žmean 20.4 g., and in P. m. bairdii from 15 to 20.9 g Ž mean 18.6 g.. Body mass differs between color-morph and sex in P. m. gracilis ŽHayssen, 1996a,b.. Approximately 30% of non-agouti females Ž and 17% of non-agouti males. weigh over 25 g, compared with only 7% of agouti females Ž and 12% of agouti males.. Non-agouti female P. m. gracilis show signs of obesity Ž 25 g. as early as 1-year-old, but obesity is not observed in P. m. bairdii Blood collection Seventeen virgin female P. m. bairdii Ž8 agouti, 9 non-agouti. and 41 virgin female P. m. gracilis Ž 20 agouti, 21 non-agouti. were sampled by retro-orbital bleeding. Blood was collected between and h, the time of day corresponding to daily peak levels of thyroxine in deer

3 J.M. Lapseritis, V. Hayssen Comparati e Biochemistry and Physiology Part A mice Ž Pitman and Bradley, At least 50 l of serum was collected from blood in two heparinized micro-hematocrit capillary tubes ŽFisher Scientific, Agawam, MA, USA, cat. no Serum was separated from cells in a capillary tube centrifuge and frozen at 20 C for 1 6 days prior to assaying Radioimmunoassays Total thyroxine Ž T. 4 was assayed by monoclonal solid phase radioimmunoassays ŽICN Pharmaceuticals, Orangeburg, NY, USA, cat. no Each sample was prepared in duplicate and mean T4 values used for statistical analysis. P. m. bairdii and P. m. gracilis were assayed separately. Independent standard curves were plotted and regression equations calculated for each assay with Microsoft Excel Statistical analysis One agouti Ž P. m. bairdii. had a low T4 value Ž 1.7 g dl. and two samples had exceptionally high T levels Ž 4 one agouti P. m. bairdii, 6.4 g dl, and one non-agouti P. m. gracilis, 4.3 g dl., and were identified as statistical outliers from box and whisker plots of each subspecies-color morph sample. Although P. m. bairdii agouti and P. m. gracilis non-agouti means were only affected by 0.03 and 0.07 g dl respectively, these three values were dropped from further analyses Žeffective n 15, n 40. P.m.b. P.m.g.. Analysis of covariance on serum thyroxine levels was conducted with SYSTAT 7.0 with subspecies, color morph, and assay as independent variables and body mass and age as covariates. Assay had no effect nor did the subspecies by color morph interaction term; thus both were dropped from the final analysis. 3. Results Both subspecies and color morph had significant effects on total thyroxine levels Ž Fig. 1.. T 4 decreased only slightly with age Ž P ; and body mass had no effect Ž P Peromyscus maniculatus bairdii of both color-morphs had significantly higher mean T4 levels than P. m. gracilis Ž P On average, T4 of P. m. bairdii was 1.9 g dl greater than that of P. m. gracilis. P. m. bairdii T4 levels ranged from 2.3 to 5.3 g dl for Fig. 1. Mean serum total thyroxine Ž T. 4 in two subspecies of agouti and non-agouti deermice Ž S.E.; n 55.. P. m. bairdii have higher T levels than P. m. gracilis Ž P , and non-agouti deer mice have higher T than agouti Ž P agoutis, and 4.1 to 6.5 g dl for non-agoutis. P. m. gracilis T4 levels ranged from 0.9 to 3.8 g dl for agoutis, and from 1.2 to 4.1 g dl for nonagoutis. Non-agouti deermice of both subspecies had significantly higher mean T4 levels than agouti Ž P , but the effect was smaller than that of subspecies. T4 of wild type agouti deermice was 0.6 g dl less than that of non-agouti animals. 4. Discussion 4.1. Subspecies differences Peromyscus maniculatus gracilis have lower thyroxine levels than P. m. bairdii. These results support much earlier studies of thyroid function in the two subspecies. Studies in the 1960s examining thyroid mass ŽEleftheriou and Zarrow, 1961., histology ŽEleftheriou and Zarrow, 1962a;. 131 Dawson, 1967, loss of I after injection of thyroid stimulating hormone ŽTSH; Eleftheriou and Zarrow, 1962b; Levy et al., 1965., or thyroid secretion rate ŽEleftheriou and Zarrow, 1962a; Dawson, all concluded that P. m. gracilis had lower levels of thyroid function than did P. m. bairdii. Eleftheriou and Zarrow Ž remarked that although P. m. gracilis is the larger of the two subspecies, P. m. bairdii has the greater thyroid activity. Thus, increased levels of thyroxine may increase metabolism and keep adult body 4

4 298 J.M. Lapseritis, V. Hayssen Comparati e Biochemistry and Physiology Part A mass low. Our results are consistent with these other measures of thyroid activity, contributing to evidence that P. m. gracilis is hypothyroid relative to P. m. bairdii. Examination of T3 and TSH levels in these subspecies, including obese individuals, may lead to further determination of thyroid effects on body mass Color-morph differences Non-agouti female deermice have higher total thyroxine levels than agouti, a pattern maintained in both subspecies. Contrary to the differences between subspecies, the larger color morph has higher T4 levels. Thus the tendency towards obesity in non-agouti female P. m. gracilis cannot be explained by hypothyroidism. The observed increased levels of thyroxine in non-agouti females should increase metabolism and decrease body mass. Lower T4 levels and increased body mass may occur for several reasons. First, female non-agouti deer mice may be insensitive to T4 negative feedback. Alternatively, increased thyroid activity may be a compensatory action for a different body mass set point caused by other factorž. s. Third, thyroxine has various developmental effects in combination with other hormones. In addition to interactions with prolactin and growth hormone, the clearance of adrenocorticoids, corticosterone binding globulin levels, and adrenal sensitivity to ACTH are altered by thyroid activity. High thyroxine levels may reduce corticosteroid levels and have a subsequent impact on metabolism ŽPitman and Bradley, 1984; Hadley, The fact that male non-agouti P. m. gracilis have nearly the same weight profile as agouti males also suggests that androgens may afford a protective value Summary In sum, Peromyscus maniculatus gracilis exhibit relative hypothyroidism in comparison with P. m. bairdii and are also a larger subspecies. In contrast, the non-agouti color morph has higher thyroid levels than wild type agouti, yet non-agouti females Ž at least in P. m. gracilis. are larger. Non-agouti females lack both agouti protein and androgens, perhaps making them more susceptible to weight gain. Over-expression of agouti is long known to cause obesity Ž Silvers 1979., but in recent years obesity research has focused on leptin, the product of the obese gene Ž Lawrence et al Leptin regulates feeding behavior and may interact with agouti or its homologue, agouti-related protein, in the hypothalamus ŽMichaud et al., 1997; Lawrence et al., In adipocytes of transgenic mice Ž Klebig et al., 1995., agouti over-expression may up-regulate leptin expression to control food intake and energy balance Ž Claycombe et al., 2000b.. The role of leptin in the non-agouti phenotype is unclear; in the absence of agouti up-regulation of leptin, orexia may contribute to weight gain, but this hypothesis has not been examined. Another unexamined hypothesis is that lack of agouti expression shifts tyrosine metabolism towards fatty acid synthesis or thyroxine imbalance, with effects on body mass. Both central Žvia hypothalamic melanocortin receptors; Fan et al., and peripheral Žvia lipid metabolism in adipose tissue; Xue and Zemel, mechanisms have been proposed for the interaction of agouti protein and the hyperphagia and obesity which occurs in conjunction with dominant mutations of the agouti locus. The data here, from animals with a recessive mutation at the locus, suggest that indirect effects on thyroid function may contribute to the abnormal weight profile. Acknowledgements The Blakeslee Trust for Genetics and Wilens Fellowship Ž Smith College. provided funding for this project. Training and P. m. bairdii samples were provided by W. Dawson and J. Crossland of the Peromyscus Genetic Stock Center, University of South Carolina, Columbia, SC. R. Thomas Zoeller at the University of Massachusetts, Amherst, generously provided technical advice and the use of a gamma counter. D. Ewell and V. Flood provided expert animal husbandry. J.M. Lapseritis conducted this work in partial fulfillment of the master s degree requirements at Smith College, Northampton, MA. References Claycombe, K.J., Wang, Y., Jones, B.H. et al., 2000a. Transcriptional regulation of the adipocyte fatty acid synthase gene by agouti: interaction with insulin. Physiol. Genomics 3,

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