YHBEH-03680; No. of pages: 11; 4C: Hormones and Behavior xxx (2014) xxx–xxx

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Review

Insulins, leptin and feeding in a population of Peromyscus leucopus (white-footed mouse) with variable fertility Jordan T. White a,b, Cori L. DeSanto a, Connie Gibbons a, Casey K. Lardner a, Andrew Panakos a, Salehin Rais a, Kathy Sharp a, Shannon D. Sullivan a,1, Wendy Tidhar a, Leanne Wright a, David Berrigan c, Paul D. Heideman a,⁎ a b c

Department of Biology, College of William and Mary, Williamsburg, VA, USA Department of Biology, Johns Hopkins University, Baltimore, MD, USA Applied Research Program, Division of Cancer Control and Population Sciences, National Cancer Institute, Bethesda, MD 20892, USA

a r t i c l e

i n f o

a b s t r a c t

Available online xxxx

This article is part of a Special Issue “Energy Balance”.

Keywords: Genetic variation Natural population Leptin Insulin Insulin-like growth factor 1 Energetics Reproduction Photoperiod Food intake Calorie adjustment

Natural populations display a variety of reproductive responses to environmental cues, but the underlying physiology that causes these responses is largely unknown. This study tested the hypothesis that heritable variation in reproductive traits can be described by heritable variation in concentrations of hormones critical to both energy balance and reproduction. To test this hypothesis, we used mouse lines derived from a wild population and selectively bred for response to short day photoperiod. Reproductive and metabolic traits of Peromyscus leucopus display heritable variation when held in short photoperiods typical of winter. Our two lines of mice have phenotypes spanning the full range of variation observed in nature in winter. We tested male and female mice for heritable variation in fasted serum concentrations of three hormones involved in energetic regulation: leptin, insulin-like growth factor 1 (IGF-1) and insulin, as well as the effects of exogenous leptin and a high energy diet on reproductive maturation. Exogenous leptin decreased food intake, but protected males from the reduction in testis mass caused by equivalent food restriction in pair-fed, saline-infused controls. A high energy diet resulted in calorie adjustment by the mice, and failed to alter reproductive phenotype. Concentrations of the three hormones did not differ significantly between selection lines but had correlations with measures of food intake, fertility, blood glucose, and/or body mass. There was evidence of interactions between reproductive traits and hormones related to energy balance and reproduction, but this study did not find evidence that variation in these hormones caused variation in reproductive phenotype. © 2014 Published by Elsevier Inc.

Contents Introduction . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . Development of selection lines . . . . Experimental design . . . . . . . . High-energy diet experiment . Leptin infusion. . . . . . . . IGF-1, insulin and leptin . . . Hormone assay quality controls Body composition . . . . . . Statistics . . . . . . . . . . Results . . . . . . . . . . . . . . . . . High-energy diet . . . . . . . . . . Leptin infusion . . . . . . . . . . .

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⁎ Corresponding author at: Department of Biology, College of William and Mary, P.O. Box 8795, Williamsburg, VA 23187-8795, USA. E-mail addresses: [email protected] (J.T. White), [email protected] (C.L. DeSanto), [email protected] (C. Gibbons), [email protected] (C.K. Lardner), [email protected] (A. Panakos), [email protected] (S. Rais), [email protected] (K. Sharp), [email protected] (S.D. Sullivan), [email protected] (W. Tidhar), [email protected] (L. Wright), [email protected] (D. Berrigan), [email protected] (P.D. Heideman). 1 Current address of S. Sullivan: Department of Medicine, Endocrine Division, Medstar Washington Hospital Center, Washington, DC, USA.

http://dx.doi.org/10.1016/j.yhbeh.2014.02.006 0018-506X/© 2014 Published by Elsevier Inc.

Please cite this article as: White, J.T., et al., Insulins, leptin and feeding in a population of Peromyscus leucopus (white-footed mouse) with variable fertility, Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.02.006

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J.T. White et al. / Hormones and Behavior xxx (2014) xxx–xxx

IGF-1. . . . . . . . . Insulin . . . . . . . . Leptin . . . . . . . . Food intake and glucose Body composition . . . Discussion . . . . . . . . Conclusion . . . . . . . . Acknowledgments . . . . . References . . . . . . . .

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Introduction Genetic variation in hormone concentrations is a potential cause of variation in life-history strategies (Hau, 2007; Williams, 2008). Studies in birds have shown that concentrations of hormones can correlate with specific life-history strategies and reproductive traits (Hau et al., 2010). Within populations of mammals, there are correlations between environmental inputs such as photoperiod and concentrations of reproductive hormones (Bartness et al., 1993; Blank and Desjardins, 1986; Heideman et al., 2010; Mintz et al., 2007). Hormonal variation has been proposed as a cause of natural variation in reproductive and life history traits (Hau, 2007; Williams, 2008). There are two sources of endocrine variation that might affect life history traits. First, a change in concentrations of circulating hormones would affect all targets of these hormones. Second, heritable variation in receptor systems or cellular responses to receptor binding would allow tissuespecific or organ system-specific responses to the same circulating hormones. Either or both could be the material upon which selection acts. Recent literature has called for new data relating heritable variation in concentrations of hormones to heritable variation in life-history strategies (Adkins-Regan, 2008; Hau, 2007; Heideman et al., 2010). A naturally variable life history trait in small mammals in the temperate zone is suppression of reproduction by short photoperiods typical of winter (Ebling and Barrett, 2008; Fournier et al., 1999; Heideman et al., 1999a; Prendergast et al., 2001). In some wild populations, there is variation in winter breeding (Carlson et al., 1989; Heideman and Bronson, 1991; Heideman et al., 1999a; Scarlett, 2004; Terman, 1993), and in some populations winter breeding is correlated with measures of food availability (Jones et al., 1998; Ostfeld et al., 1996; Wolff, 1996), but not in others (Scarlett, 2004). In many populations, short photoperiod may fail to suppress reproduction in some individuals, may suppress reproduction completely in others, and may suppress reproduction to an intermediate level in others, with significant heritability to this variation (Blank and Desjardins, 1986; Heideman and Bronson, 1991; Heideman et al., 1999a; Prendergast et al., 2001). This heritable variation in winter reproduction, a life history trait, may be the consequence of a life history trade-off (Heideman et al., 2005). The harsh conditions of winter may create a selective disadvantage for reproduction because of the added costs of thermoregulation and foraging (Bronson, 2009; Hill et al., 2008; Prendergast et al., 2001), including energetic costs and risk costs. However, if resources are abundant and risks low for some individuals in a population, then winter reproduction may be favored. In Peromyscus leucopus, conditions favoring either winter reproduction or winter infertility are temporally and spatially variable (Lynch, 1973; Lynch et al., 1981), resulting in variable selection on the intensity of winter reproductive suppression (Avigdor et al., 2005). Physiological factors that may regulate the intensity of winter reproductive suppression include hormones that either alter or respond to food intake, energy balance, and energy reserves. Short photoperiod can affect food intake, body mass, body composition, metabolic rate, and other non-reproductive traits (Bartness and Wade, 1985; Heideman et al., 2005; Kaseloo et al., 2012; Morgan et al., 2006). Conversely, food intake and body condition can alter reproductive responses to short and intermediate photoperiods (Heideman et al., 1998; Paul

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et al., 2009; Reilly et al., 2006). These results imply that in nature, the physiological outcome of fertility or infertility in winter results from a complex interaction of reproductive hormones, energetic hormones, environment, diet, and ingestive behavior (Schneider et al., 2013). Few, if any, studies have investigated heritable variation in hormones of reproduction and energy balance in a natural population. We have used a model in which natural genetic variation for winter fertility has been concentrated in two selectively bred lines from a naturally-variable wild population of white-footed mice, P. leucopus. In this model, lines were formed for either strong inhibition of reproduction in short photoperiod: reproductively responsive to short photoperiod (responsive, R), versus low or no inhibition of reproduction in short photoperiod (nonresponsive, NR), with a control (C) line maintained without selection. During 10 generations of selection, the NR and R lines shifted their distribution of phenotypes in short photoperiod toward the extremes of the wild population in winter (Heideman, 2004; Heideman and Pittman, 2009; Heideman et al., 1999a). The selection lines have substantial heritable differences between selection lines in gonadal development, reproductive accessory glands, GnRH neurons, and luteinizing hormone (Table 1). The differences between lines are not due to malfunction of the circadian system (Majoy and Heideman, 2000), and the selection lines both respond to melatonin and undergo similar reductions in body mass in short photoperiod (Table 1). Interestingly, the two lines have also shown heritable differences in metabolic rate, feeding, and activity. This suggests that these selection lines might provide a model to examine natural variation in relationships among body composition, food intake, and hormones of energetics in relation to winter reproduction. Here, we test for differences between selection lines as well as correlations among concentrations of hormones and food intake, body mass, and reproductive traits. This study addressed three specific questions: (1) Can the photoperiod response be phenotypically plastic? (2) Can an energy-related hormone, leptin, cause reproductive phenotypic differences and are differences in three energy-related hormones heritable? (3) Is body composition, and particularly fat mass, related to the response to short photoperiod? In relation to the first question, reduced access to food suppresses reproduction in NR mice in SD, but not LD (Reilly et al., 2006). Thus, we tested the converse hypothesis that an abundance of high energy food should stimulate reproduction in R mice in SD. In order to answer this first question we examined the reproductive phenotype and food intake of R males in response to a highly palatable and nutritious high calorie diet in a short day photoperiod. Second, we tested the hypothesis that variation in concentrations of hormones related to feeding and/or metabolism could cause differences in reproduction in short photoperiod. To answer our second question, in separate short photoperiod experiments we administered leptin to R line mice and quantified basal, fasted concentrations of three hormones in mice from the two selection lines: insulin-like growth factor, leptin, and insulin. Insulin-like growth factor-1 and leptin have effects on feeding and reproduction (insulin-like growth factor-1: Daftary and Gore, 2005; Veldhuis et al., 2006; Ross et al., 2009, leptin: Clarke and Henry, 1999; Kohno et al., 2007). Insulin is involved in glucose homeostasis and metabolism (Hadley and Levine, 2007).

Please cite this article as: White, J.T., et al., Insulins, leptin and feeding in a population of Peromyscus leucopus (white-footed mouse) with variable fertility, Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.02.006

J.T. White et al. / Hormones and Behavior xxx (2014) xxx–xxx

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Table 1 Summary of differences between selection lines selected for reproductive maintenance (nonresponsive to short photoperiod, NR) or reproductive suppression (responsive to short photoperiod, R) in short, winter-typical photoperiods (L8:D16). Traits

Selection Lines

Photoperiod

Sex

Citations

Reproductive traits Gonads and reproductive accessory organs Neurons or hormones of the reproductive axis (GnRH neurons and LH) Correlations between food intake and reproductive measures

NR N R NR N R Both lines

SD & LD SD & LD Correlated in SD; not correlated in LD

M&F M or F M

1, 2, 3, 4, 5, 6, 7, 8, 9, 10 5, 8 4, 7, 9, 10

Body mass, feeding, activity, and metabolism Body mass (12°, 22°, or 28 °C) Food intake (12°, 22°, or 28 °C) Activity (12°, 22°, or 28 °C) Metabolic rate (12°, 22°, or 28 °C)

NR NR NR NR

SD & LD SD & LD SD & LD SD, not LD

M&F M M M

1, 2, 3, 4, 5, 6, 7, 8, 9, 10 4, 7, 9, 10 4, 9, 10 9, 10

Other responses to SD or melatonin Lower body mass and food intake in SD than in LD Hypothalamic neuronal firing Rate in response to melatonin in vitro Iodomelatonin binding (in the MPOA and BNST)

NR = R NR = R NR N R

LD vs SD LD vs SD SD

M F M

3, 5, 9, 19 6 2

= R or NR N R N R or NR = R N R or NR = R NR

(1) Heideman et al. (1999a), (2) Heideman et al. (1999b), (3) Majoy and Heideman (2000), (4) Heideman et al. (2005), (5) Avigdor et al. (2005), (6) Fetsch et al. (2006), (7) Reilly et al. (2006), (8) Heideman et al. (2010), (9) Kaseloo et al. (2012), and (10) Kaseloo et al. (in press) (abbreviations: MPOA: medial preoptic area; BNST: bed nucleus of the stria terminalis).

Finally, if the concentration of leptin is a cause of variation in reproduction in short days between the NR and R selection lines, then we predicted differences in body fat levels and body composition between the selection lines. Thus, in this paper we report on feeding behavior, hormones and body composition in relation to variation in fertility. Methods Development of selection lines The selection lines of P. leucopus were initiated in 1995 from a wild population near Williamsburg, VA (latitude 37 × 3°N, longitude 76 × 7°W) (Heideman et al., 1999a). A parental laboratory generation was used to found three lines: one control line not bred for a photoperiod response and two lines bred for either a photoperiod response or the lack of a photoperiod response (Heideman et al., 2010). Our wildsource population is housed at the College of William and Mary Population Laboratory in two photoperiods: short day (SD, 8L:16D) and long day (LD, 16L:8D). Mice from a parental generation were raised in SD, examined at 70 ± 3 days of age and assessed for reproductive phenotype based on the size of gonads and, for females, also the diameter of the uterus and presence or absence of macroscopically visible mature follicles or corpora lutea. A female was considered reproductively inhibited by SD if the ovary examined was ≤2 mm in length, if the ovary lacked visible follicles or corpora lutea, and if the diameter of the uterine cornua was ≤0.5 mm. A female was considered nonresponsive to SD if the ovaries were large (usually N 3.5 mm in length), large visible follicles or corpora lutea, and a uterine diameter N 1 mm. For males, a testis size measure (length × width of testis) was used to classify males as either R (b 24 mm2) or NR (N 32 mm2). R mice were paired with R mates and NR mice were paired with NR mates to found the R and NR lines, respectively. Selective breeding was continued for the subsequent 10 generations, after which selection was relaxed. Mice from the parental generation were chosen and paired randomly to form the unselected control line, which was maintained without selective breeding (Heideman et al., 1999a). Additional details about the selection lines are available elsewhere (Heideman, 2004; Heideman and Pittman, 2009; Heideman et al., 1999a). Experimental design High-energy diet experiment In this experiment, we tested the hypothesis that energy availability and day length interact to influence reproductive success. We predicted that a highly palatable high-energy diet would enhance reproductive function in responsive (R) mice in SD or LD. At age 27 ± 3 days, male mice from the R and control (C) lines were divided into eight groups matched for body weight at weaning. Four groups,

consisting of R mice in short day or long day photoperiods (N = 7, N = 8 respectively), and C mice in SD or LD (N = 7, N = 8) were fed a high-energy diet (45 kcal% fat, 35 kcal% carbohydrate, 20 kcal% protein, 4.7 kcal/g, Open Source Diets), with supplemental Eagle Brand sweetened condensed milk (3.3 kcal/g) from a sipper bottle, both ad libitum, for 8 weeks. In a pilot experiment, this diet was palatable and highly preferred by our mice. Four control groups composed of C mice in SD and LD (N = 7, N = 8) and R mice in SD and LD (N = 7, N = 7) were fed a standard rodent diet (10 kcal% fat, 70 kcal% carbohydrate, 20 kcal% protein, 3.8 kcal/g, ad libitum). Food in food hoppers was weighed weekly. After eight weeks of treatment, the mice were euthanized using gaseous CO2, weighed, and tested for reproductive development. Measurements in this experiment were taken blind with respect to treatment. Paired testes were removed and immediately weighed. Paired seminal vesicles were removed, stripped of fluid, and weighed.

Leptin infusion Mice were infused with leptin using Alzet miniosmotic pumps (model 2004, flow rate 0.25 μL/h for 28 days; Alza Corporation, Palo Alto, CA) to provide a constant release of leptin over four weeks. Recombinant mouse leptin was generously provided by Amgen Inc. (Thousand Oaks, CA). Male mice were divided into three groups matched for body weight, testis index, and mouse line (R or C) at age 70 ± 3 days. Food intake over the 4–6 day pretreatment period was compared with mean food intake over 5-day periods during treatments. Mice in the leptin group (N = 7) were anesthetized with isofluorane and implanted s.c. with an Alzet osmotic pump that provided a constant infusion of 20 μg/day of leptin in saline with 0.1% BSA. This dose was chosen because previous studies in rodents reported significant reproductive responses using similar doses (Barash et al., 1996; Chehab et al., 1997). Pair fed controls (N = 7) were implanted with a pump that delivered only the saline with BSA; and mice in a second control group (N = 5) were fed ad lib and also implanted with a pump that delivered only saline with BSA. To control for the effect of reduced food intake caused by leptin, each mouse in the saline control group was pair-fed with a mouse of the same body weight in the leptin-treated group. Pairs were established by matching food intake of mice during the pre-treatment measures of food intake. Mice in the control group received the amount of food eaten on the previous day by their weight-matched pair mate in the leptin treatment group. Pair-fed control mice ate all of the food offered on 97% of days. After four weeks of treatment, mice were euthanized using gaseous CO2 and their body, testes, and fluid-stripped seminal vesicles were weighed. A preliminary experiment with a treatment of daily peripheral leptin injection gave similar results and is not reported here.

Please cite this article as: White, J.T., et al., Insulins, leptin and feeding in a population of Peromyscus leucopus (white-footed mouse) with variable fertility, Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.02.006

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J.T. White et al. / Hormones and Behavior xxx (2014) xxx–xxx

IGF-1, insulin and leptin Sample size for the IGF-1 assay was 12 males and 12 females from each line, all held in SD (N = 48). Sample size for the leptin assay was 17 R males, 14 R females, 16 NR males, and 15 NR females (N = 62) in SD. Sample size for the insulin assay was 9 R males, 11 NR males and 12 females of each line (N = 44) in SD. Mice were selected at random and only mice between 7 and 16 weeks old were used. Food and water were provided ad libitum. Mice were caged separately and food in the hopper of each cage was weighed at one-week intervals for three weeks. To reduce inaccurate measures of food intake, mice that had chewed food that had fallen onto the cage floor were excluded from the analyses (final N = 152). Body mass and food intake were measured on the same days. Mice were culled at the end of the three-week period, although food intake data was only collected for a two-week period. On the day of culling for hormone assay, mice were fasted starting at 900 h (lights on) for the 6 h before blood collection to reduce variation in feeding related hormones. This population of mice eats about 93% of its total food intake during the dark phase, thus the last meal of each mouse was at least six hours prior to blood collection. At the time of blood collection (1500 h), each mouse was taken quietly to a separate room and individually anesthetized with isofluorane. While under deep anesthesia, each mouse was euthanized and trunk blood was collected. One drop of blood was used to measure glucose (N = 139) with a OneTouch Ultra glucometer, which can measure concentration of glucose from 20 to 600 mg/dL. Glucometer accuracy was tested using a control solution. All collections occurred between 1500 and 1700 h, which is immediately before the night phase for SD mice (1700 h). After collection, blood was allowed to clot for about 30 min at 4 °C, subsequently centrifuged, and serum was collected. Serum that showed signs of contamination with dislodged or suspended blood cells was respun, and hemolyzed serum was not used in the assays. After collection, serum was frozen at − 70 °C for later use. Immediately after blood collection, mice were dissected, and paired testes, paired fluid-stripped seminal vesicles, paired ovaries, and uteri were removed and weighed.

a

IGF-1 was assayed with an R&D Systems Quantikine Mouse IGF-1 kit (Cat. MG-100). The assay had an intra-assay coefficient of variation of 5.6%, 3.3%, and 4.1% for concentrations at 82, 269, and 921 pg/mL, respectively. The range for the assay standard curve was 31.2 pg/mL to 2000 pg/mL. Prior to the assay, serum was diluted 500 fold, and the concentrations reported in the results have been corrected for the dilution factor. All reagents were used within the parameters set by the assay manual. The reconstituted standard was used on the same day. Samples were run in singlet. Insulin was assayed with a Millipore ELISA Rat/Mouse Insulin kit (Cat. EZRMI-13 K). The assay had an intra-assay coefficient of variation of 8.35%, 0.92%, and 1.92% for concentrations at 0.32, 1.69, and 3.45 ng/mL, respectively. The range for the assay standard curve was 0.2 ng/mL to 10 ng/mL. All reagents were used within the parameters set by the assay manual. Samples were run in singlet. Leptin was assayed using two Millipore ELISA Mouse Leptin kits (Cat. EZML-82 K). The range for the assay standard curve was 0.2 ng/mL to 30 ng/mL. During our analysis, we assigned 0.2 ng/mL to all samples that had a concentration below the standard curve. The first assay used 46 mice and the second assay used 44 mice with 26 samples repeated from the first assay. Two values extrapolated by the software were excluded (final N = 62). In both assays, 27 of the samples had a concentration of leptin below the standard curve. The average inter-assay coefficient of variation between the two runs was 13.1%, and the average intra-assay coefficient of variation between the two runs was 6.1%. Given these coefficients of variation, we accepted the results of both assays and averaged the concentrations of leptin for the samples that were run twice. The IGF-1, insulin and leptin assay readings were conducted by a BioTek ELx800 Microplate Reader. The IGF-1 assay was read at 450 nm with a correction filter of 540 nm. The insulin assay and both leptin assays were read at 450 nm with a correction filter of 590 nm. Each standard curve was built using a 4-P fit calculated by the plate reader.

b

35 30

NS 0.3

25

Body Weight (g)

0.4

NS

20

Testes 0.2 Weight (g)

15 10

0.1

5 0

St LD

St SD

HE LD

0.0

HE SD

c

St LD

St SD

HE LD

HE SD

d 0.8

0.08

NS

NS 0.6

0.06

Daily Caloric 0.4 Intake (kcal/g Body mass) 0.2

Seminal Vesicles 0.04 Weight (g) 0.02

0.00

St LD

St SD

HE LD

HE SD

0.0

St

HE

Fig. 1. Caloric intake, body mass, and reproductive measures after eight weeks of a control, standard diet (St) or highly palatable, high-energy (HE) diet in long photoperiod (LD: 16L:8D) or short photoperiod (SD: 8L:16D). Data from the responsive and control lines were combined because there were no differences due to line. (a) body mass, (b) wet mass of paired testes, (c) wet mass of fluid-stripped seminal vesicles, (d) daily caloric intake (means ± 95% Confidence Intervals). Sample sizes: St-LD = 14, St-SD = 13, HE-LD = 16, HE-SD = 13.

Please cite this article as: White, J.T., et al., Insulins, leptin and feeding in a population of Peromyscus leucopus (white-footed mouse) with variable fertility, Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.02.006

J.T. White et al. / Hormones and Behavior xxx (2014) xxx–xxx

Hormone assay quality controls The assay for IGF-1 had a standard curve with r = 1.000. The three quality controls were valid. Two NR females had concentrations of IGF-1 that were above the standard curve and were assigned the highest values detectable by the curve. The assay for insulin had a standard curve with r = 0.982 and both quality controls were valid. Each assay for leptin had r = 0.987, and the two quality controls in each of the two assays were valid. Out of 62 mice, 35 had concentrations of leptin below the standard curve of the assay and were assigned a concentration of 0.2 ng/mL. Body composition In a separate experiment, we measured the body composition of male mice from each line and photoperiod (sample sizes: R in SD = 11, R in LD = 16, NR in SD = 13, NR in LD = 17). To determine fat weight, lean weight, bone mineral density (BMD), and bone mineral content (BMC), we used dual-energy X-ray absorptiometry (DXA) (GE Lunar Piximus II, Madison, WI, USA). Mouse carcasses were repositioned on the specimen tray after each individual scan. GEsupplied software was used to extract data from the scans. Animals were weighed before and after necropsy and before scanning. In order to calculate an adjusted fat weight, the fat weight estimated for the necropsied carcasses and the fat weight in tissue removed during necropsy were added together. Lean weight was then defined as weight prior to necropsy minus the adjusted fat weight. The bone density in the tibia alone and in the vertebrae alone were estimated by making the region of interest either the left tibia or the vertebrae from the most medial to the most lateral point. After selection of these regions of interest, bone mineral density (g/cm2) and bone mineral contents (g) were estimated based on radiation energy per pixel. This method has been validated using gravimetric and chemical extraction (Berrigan et al., 2005).

a 4

Ave. Daily Food Cons. (g)

Leptin Saline P = 0.02

3

Statistics For the leptin infusion and body composition experiments, means and standard errors of the means are presented for gonadal measurements and body weights (JMP statistical package, Version 3.1, SAS Institute Inc., Cary, NC). For the leptin infusion experiment, food intake and effects of photoperiod on gonadal development were analyzed by repeated measures ANOVA and ANOVA, respectively. Body composition was analyzed by ANOVA. Statistics of the high-energy diet, hormone, and glucose studies were calculated using R version 3.0.1 (R-CoreTeam, 2013) by Type III SS ANOVA. Siblings were included in some data sets, but the effects of family were not significant in preliminary analyses, and family was not included as a variable in the final analyses. Variances were significantly different among treatments for paired seminal vesicle mass, and these data were log transformed before analysis. To analyze concentrations of leptin and insulin, body weight was related to hormone concentrations, and non-parametric ANCOVA (Weight × Gender × Line) was used to include body weight. Nonparametric ANOVA (Gender × Line) results are presented for IGF-1. Following ANOVA, the Tukey–Kramer test was used to determine the significance of multiple comparisons. A type 1 error of 5% (α = 0.05) was used as a cut off value for significance of all statistics. Experiments were reviewed and approved by the institutional animal care and use committee of The College of William and Mary and meet National Institutes of Health requirements. Results High-energy diet We sought to determine if a high-energy diet would reverse the effects of a SD photoperiod on the responsive (R) selection line or unselected control (C) line. Eight weeks of a high-energy diet did not affect

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Fig. 2. Mean (±SEM) of average daily food consumption (a), body mass (b), paired testis mass (c), and paired seminal vesicle mass (d) of photoresponsive mice in SD given daily injections of leptin or saline vehicle, with both groups matched for food intake. In (a), the attained level of statistical significance is given for a comparison of food intake in the leptin treatment group and ad lib fed control group. In b, c, and d, attained level of significance is given for statistical comparisons of means using ANOVA. NS indicates lack of statistical significance.

Please cite this article as: White, J.T., et al., Insulins, leptin and feeding in a population of Peromyscus leucopus (white-footed mouse) with variable fertility, Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.02.006

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body mass (P N 0.40, Fig. 1a) or overcome the reproductive effects of a short photoperiod on photoresponsive mice. The C and R lines did not differ in body mass (P N 0.20), but with just photoperiod considered, mice in SD had significantly lower body mass than mice in LD (P b 0.05). Seminal vesicle and testes data were compared using ANCOVA, with line, photoperiod, and diet as factors and body mass as a covariate. The high-energy diet did not have a significant effect on either testes mass (P N 0.30, Fig. 1b) or seminal vesicle mass (P N 0.70, Fig. 1c). For testes mass, there were significant effects of the covariate (body mass; P b 0.001) and two of the factors (line and photoperiod; P b 0.01, P b 0.03 respectively). For seminal vesicles mass, there was a marginally insignificant effect of the covariate (body mass; P = 0.055), a significant effect of photoperiod (P b 0.02), but not a significant effect of line (P N 0.14). Caloric intake per day was not correlated with initial body mass measured at week 0 (R = 0.08; P N 0.50). However, there was a strong correlation between caloric intake throughout the experiment and body mass measured at week 8 (R = 0.68; P b 0.001). An ANCOVA on average daily calorie consumption with mass as the covariate revealed a significant effect of body mass (P b 0.0001), but there were no significant effects on average daily caloric intake from photoperiod (P N 0.30), line (P N 0.50), or interactions (P N 0.10 for all). Most importantly, the high-energy diet treatment had no significant effect on caloric intake (P N 0.48, Fig. 1d). This experiment had a statistical power of 0.8 (i.e., 0.8 probability) of detecting the following minimum differences

a

between the means of the groups: N0.17 kcal/g/day, N0.10 g testes mass, and N0.017 g seminal vesicles mass. Leptin infusion Leptin-infused R mice ate 10% less than the ad lib, saline-infused controls (repeated measures ANOVA, F = 3.73, P = 0.021, Fig. 2a). Four weeks of leptin infusion caused no significant differences in the body masses of the groups (F = 1.12, P = 0.35, Fig. 2b). Testis mass of the leptin treatment group was similar to that of the ad lib fed controls, but both of these groups had a larger mean testis mass than did the pair-fed salineinfused controls (F = 4.55, P = 0.027; Fig. 2c). A post-hoc contrast comparison indicated that testes mass of the pair-fed saline-control group was significantly less than that of the other two groups (F = 9.10, P = 0.008). There was a statistical trend for differences in seminal vesicle mass (F = 3.16, P = 0.07; Fig. 2d). A post-hoc contrast comparison indicated that seminal vesicle mass of the pair-fed saline control group was significantly less than that of the other two groups (F = 6.12, P = 0.025). IGF-1 Concentration of IGF-1 in fasting serum did not differ significantly between any of the groups tested (gender × mouse line) (P N 0.10, Fig. 3a). IGF-1 was correlated positively with reproductive measures in

700 600

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Fig. 3. Serum concentrations of insulin-like growth factor 1 (IGF-1) in short photoperiod in relation to selection line, gender, and reproductive organ mass. (a) Concentrations of IGF-1 according selection line (nonresponsive, NR, and responsive, R) and sex (male, M, and female, F) (Means ± 95% Confidence Interval). (b) Correlation between mass of fluid-stripped seminal vesicles and concentrations of IGF-1 in NR males (open circles, dashed line, r = 0.59, P b 0.05) and R males (closed circles, solid line, r = 0.48, P b 0.07). (c) Correlation between mass of paired testes and concentrations of IGF-1 in NR males (open circles, dashed line, r = 0.75, P b 0.01) and R males (closed circles, solid line, r = 0.64, P b 0.02). (d) Correlation between mass of paired ovaries and concentrations of IGF-1 in females from both lines (NR = open circles and R = closed circles, r = 0.45, P b 0.02). (e) Correlation of uterine mass and concentrations of IGF-1 in females from both lines (NR = open circles and R = closed circles, r = 0.78, P b 0.001).

Please cite this article as: White, J.T., et al., Insulins, leptin and feeding in a population of Peromyscus leucopus (white-footed mouse) with variable fertility, Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.02.006

J.T. White et al. / Hormones and Behavior xxx (2014) xxx–xxx

7

males and females of both lines. The mass of seminal vesicles was correlated with the concentration of IGF-1; within groups only the correlation for NR was significant (r = 0.59, P b 0.05, Fig. 3b). In R and NR males, IGF-1 was correlated linearly with paired testes mass (r = 0.75 and r = 0.64, respectively, both P b 0.05, Fig. 3c). In R and NR females, IGF-1 was correlated with paired ovary mass and uterine mass (r = 0.45, P b 0.02, Fig. 3d and r = 0.78, P b 0.001, Fig. 3e respectively). IGF-1 was correlated with body mass in R females (r = 0.71, P b 0.01) but not in any other group or overall. IGF-1 concentration was correlated with glucose and food intake in the mice as a whole (r = 0.27, P b 0.05 and r = 0.34, P b 0.02, respectively).

female groups (P b 0.02, Fig. 5b) and trended towards a higher body mass than R males (P b 0.06). Therefore, we used ANCOVA to analyze the concentration of leptin between the groups. There were no significant differences between the NR and R groups (Fig. 5c). Besides weight (P b 0.001), the only significant effect on leptin was the interaction of gender and weight (P b 0.01). Concentration of leptin also correlated with weekly food intake (r = 0.25, P b 0.03, Fig. 5d), but not with blood glucose, mass of uteri, paired testes, paired seminal vesicles, or paired ovaries.

Insulin Concentration of insulin in fasting serum did not differ significantly between the R and NR lines (P N 0.10, Fig. 4a). Concentration of insulin was correlated positively with body mass (r = 0.85, P b 0.001, Fig. 4b), blood glucose (r = 0.33, P b 0.02, Fig. 4c), and food intake (r = 0.45, P b 0.01, Fig. 4d) considering the mice as a whole. The NR males and R males weighed more than the R females (non-parametric ANOVA and Tukey HSD, P b 0.05). Insulin did not correlate with the mass of paired testes, seminal vesicles, paired ovaries or uteri.

It was predicted that the NR line would require a greater amount of food than the R line because maintaining their reproductive abilities in SD comes at an energetic cost to the NR mice (Kaseloo et al., 2012). The only significant difference among groups was between NR males and R females (P b 0.05, Fig. 6a), but there was no overall effect of selection line. There was no significant difference in concentration of blood glucose between selection lines (P N 0.50, Fig. 6b). Both NR and R mice had an average concentration of glucose of about 87 mg/dL (4.8 mM). Testis mass and seminal vesicle mass were not significantly correlated with concentration of glucose (P N 0.1 for both).

Leptin

Body composition

Concentration of leptin in fasting serum was correlated with body mass (R = 0.87, P b 0.001, Fig. 5a), which raised the possibility that body mass would act as a significant covariate in our analyses. Furthermore, the NR males had a significantly higher body mass than the

Line and photoperiod had significant effects on total body mass, lean body mass, and bone mineral content, and photoperiod affected bone mineral density significantly, while fat body mass did not vary significantly (Tables 2 and 3). NR mice were heavier than R mice, and mice

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Fig. 4. Serum concentrations of insulin in short photoperiod in relation to selection line, gender, body mass, blood glucose, and food intake. In b, c, and d, sexes and lines were combined because there were no differences by sex and line. Abbreviations are as in Fig. 3; sample sizes: 12 NR F, 11 NR M, 12 R F, 9 R M. (a) Concentrations of insulin according selection line (nonresponsive, NR, and responsive, R) and sex (male, M, and female, F) (means ± 95% confidence Interval). (b) Correlation between body mass and concentrations of insulin (r = 0.85, P b 0.001). (c) Correlation between blood glucose and concentrations of insulin (r = 0.33, P b 0.02). (d) Correlation between weekly food intake and concentrations of insulin (r = 0.45, P b 0.01).

Please cite this article as: White, J.T., et al., Insulins, leptin and feeding in a population of Peromyscus leucopus (white-footed mouse) with variable fertility, Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.02.006

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J.T. White et al. / Hormones and Behavior xxx (2014) xxx–xxx

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Leptin (ng/mL) Fig. 5. Serum concentrations of leptin in short photoperiod in relation to selection line, gender, body mass, and food intake. Abbreviations are as in Fig. 3; sample sizes: 15 NR F, 16 NR M, 14 R F, 17 R M. (a) Correlation between body weight and concentrations of leptin for all mice combined (r = 0.87, P b 0.001). When data were subdivided by line and gender, only correlations for male mice were statistically significant (both r N 0.77, P b 0.001). (b) Body mass in relation to line and gender in mice assayed for serum leptin (means ± 95% confidence interval). (c) Serum concentrations of leptin in relation to line and gender (means ± 95% confidence interval). (d) Correlation between weekly food intake and concentrations of leptin (r = 0.26, P b 0.03).

in LD were heavier than mice in SD, with no significant interaction of photoperiod and line (Tables 2 and 3). The difference in body mass was due largely to differences in lean body mass, and not to differences in fat mass (Table 2). Bone mineral content co-varied with body mass and lean body mass, with higher mineral content in NR than R mice, and higher mineral content in LD than in SD, with no statistically significant interaction (Tables 2 and 3). Bone mineral density was slightly lower in a SD photoperiod (Tables 2 and 3). Discussion Our main findings are that heritable differences in photoresponsiveness were not accounted for by heritable differences in fasting serum concentrations of leptin, insulin, or IGF-1 measured at time 1500 h. Furthermore, mice selectively bred to vary in reproductive photoresponsiveness did not differ in their reproductive or ingestive behavior response to leptin or to high-calorie diets. Thus, heritable variation in winter reproduction is apparently not caused by heritable differences in these additional factors that could have broad pleiotropic effects: feeding behavior, a metabolic fuel (glucose), amount of body fat, leptin, IGF-1, or insulin. Attempts to manipulate energy intake with a preferred, high energy diet resulted only in calorie adjustment, not reproductive modulation. This is consistent with the hypothesis that heritable variation in winter reproduction might be limited to variation within the reproductive axis, rather than variation in aspects of energy balance. These results have implications for reproductive and life history variation that is important in ecology and human biology. Two leading hypotheses for causes of endocrine variation propose that biologically important variation may be (1) in concentrations of circulating hormones or, alternatively, (2) in specific targets of those hormones (including variation in receptor expression and the numbers and

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Fig. 6. Food intake and blood glucose in short photoperiod in relation to line and gender (means ± 95% confidence intervals). Abbreviations are as in Fig. 3. (a) Food intake (Sample sizes: 45 NR F, 38 NR M, 37 R F, 32 R M; P b 0.05 between NR M and R F). (b) Blood glucose (Sample sizes: 41 NR F, 39 NR M, 33 R F, 26 R M.).

Please cite this article as: White, J.T., et al., Insulins, leptin and feeding in a population of Peromyscus leucopus (white-footed mouse) with variable fertility, Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.02.006

J.T. White et al. / Hormones and Behavior xxx (2014) xxx–xxx

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Table 2 Body composition in mice from the Nonresponsive (NR) and Responsive (R) selection lines in long (LD) and short photoperiods (SD) (mean ± SEM). Line

NR

Photoperiod

LD

Body mass Lean body mass Fat body mass Bone mineral density Bone mineral content

21.0 17.2 3.5 0.041 0.324

R SD ± ± ± ± ±

0.6 0.4 0.3 0.005 0.009

20.2 15.9 4.0 0.040 0.309

activity of target cells). Some studies have associated genetic variation with different concentrations of these hormones in humans, laboratory rats, and ewes (Afolayan and Fogarty, 2008; Hegele et al., 2000; Mitchell et al., 1996, 2006), but it has not been known how such variation in hormones related to growth and energy balance might be related to variation in reproduction in natural populations. Because hormonal signaling indicating adequate energy balance and energy reserves is required for maturation and fertility (Schneider et al., 2013), heritably high and low concentrations of energetic hormones might cause heritable variation in the reproductive axis. We predicted that selection lines that have differed in feeding and the reproductive response to food would also differ in circulating concentrations of hormones that may link energetics to reproduction: leptin, IGF-1, and insulin. Reproductive responses to a high-energy diet would further support a link between feeding and variation in reproduction. Similarly, heritable variation in body fat would support a link to energy stores and reproduction. We found that leptin infusion could slightly alter reproductive responses to short photoperiod. However, a highly palatable, high-energy diet did not affect either caloric intake or reproduction. There was no evidence for heritable differences between selection lines in percent body fat. A previous finding of differences in food intake between selection lines (Heideman et al., 2005; Kaseloo et al., 2012) was not replicated in this study. Most importantly, there was no evidence for heritable differences in concentrations of leptin, IGF-1, and insulin in relation to reproductive phenotype. In our high-energy diet treatment, calorie adjustment can explain the lack of weight gain in Peromyscus fed an ad libitum high-energy diet (Fig. 1). Deer mice (Peromyscus maniculatus), a close relative of P. leucopus, have been shown to maintain their body weight when given a diet of varying fat content for 30 days. Deer mice that consumed a higher energy diet decreased food intake and maintained stable body weight (Mawhinney and Miller, 1990). Unlike many strains of laboratory mice and rats, highly palatable food with high energy content did not result in changes in caloric intake in deer mice or our mice. Without changes in caloric intake, an absence of change in reproductive phenotype is unsurprising (Fig. 1). This suggests that mice in our R line are obligately suppressed reproductively by SD, with reproductive inhibition remaining unaltered by increases in palatability or energy density of food. In contrast, mice in the NR line are phenotypically plastic in SD based upon the reliability of food availability (Reilly et al., 2006). Paul and coworkers (Paul et al., 2009) found a similar effect in Siberian

Table 3 ANOVA table for body composition measurements of mice from the Nonresponsive and Responsive selection lines in long and short photoperiods. Columns indicate the F statistics and achieved probability value for the effects of selection line, photoperiod, and interaction between line and photoperiod. Effects that were significant at p b 0.05 are indicated in bold font. Line

Body mass Lean body mass Fat body mass Bone mineral density Bone mineral content

Photoperiod

Interaction

F

P

F

P

F

P

5.98 19.40 0.002 2.45 7.47

0.02 0.0001 0.98 0.12 0.009

7.11 22.62 0.02 9.33 5.96

0.01 0.0001 0.94 0.004 0.02

2.79 3.22 1.27 1.30 1.26

0.10 0.08 0.26 0.26 0.27

LD ± ± ± ± ±

1.1 0.5 0.7 0.009 0.014

20.4 16.0 4.0 0.041 0.306

SD ± ± ± ± ±

0.8 0.4 0.5 0.006 0.010

16.7 13.0 3.4 0.038 0.267

± ± ± ± ±

0.8 0.4 0.5 0.008 0.010

hamsters (Phodopus sungorus), in which a restricted diet inhibited reproduction in intermediate photoperiods. Differences between our mouse strains in the reproductive effects of food intake could be due to endocrine variation. If variation in the circulating concentration of leptin is a possible cause of differences between selection lines, then supplemental leptin should overcome reproductive suppression in SD, at least partially. Overall, the effect of leptin induced a redistribution of metabolic priorities. Decreased food intake decreases resources available for reproduction, but even though leptin-infused mice ate significantly less food than saline-treated adlib fed controls, gonadal mass was maintained above pair-fed controls (Fig. 2). Thus, exogenous leptin can have statistically significant effects on both food intake and reproductive phenotype in white-footed mice, as expected based on results on other species (Schneider et al., 2013). The correlations observed between the concentration of IGF-1 and masses of reproductive organs (Fig. 3) suggest that the concentration of IGF-1 is related to reproductive development in our population of P. leucopus, consistent with findings on other species (Daftary and Gore, 2005; Todd et al., 2007; Villalpando et al., 2008). However, despite positive correlations between body mass and insulin as well as leptin (Figs. 4, 5), we found no significant correlations of insulin or leptin with the mass of gonads or reproductive accessory organs. This suggests that neither the baseline concentration of insulin nor the baseline concentration of leptin is related to the reproductive traits investigated in this study, even though endogenous leptin can be protective of reproductive development during food restriction (Fig. 2). Concentration of insulin was correlated with concentration of glucose, as expected, but blood glucose did not differ between selection lines (Fig. 6). While body mass differed between selection lines in this study, unlike most previous experiments on these lines (Avigdor et al., 2005; Heideman et al., 1999a, 1999b, 2005, 2010), percent body fat did not differ between selection lines (Tables 2, 3). This study did not replicate a finding from earlier studies (Heideman et al., 2005; Kaseloo et al., 2012) that showed higher food intake in the NR than R selection lines, perhaps because the current study was conducted after selection was relaxed. Most importantly, we found no evidence supporting the hypothesis that heritable variation in reproductive phenotype might be due to heritable variation in serum concentrations of leptin, IGF-1, or insulin (Figs. 3, 4, 5). Where heritable variation exists in a system such as the reproductive axis, the cause of that variation might be (1) hormones and cells specifically within that system, or (2) the response to hormones, metabolic fuels, and other indicators of body condition or environment. Variation in concentrations of energetic hormones or metabolic fuels would have multiple effects on many other systems (Schneider et al., 2013). This study suggests that three hormones with broad effects on physiology and behavior, leptin, IGF-1, and insulin, are apparently not an important cause of heritable variation in the reproductive axis in SD in this population. Correlations between reproduction with metabolism and food intake in this population (Heideman et al., 2005; Kaseloo et al., 2012; Reilly et al., 2006) may be caused by reallocation of metabolic priorities by the reproductive axis. It is important to note that any or all three of the hormones we measured could be involved in reproductive modulation in white-footed mice without being a cause of heritable variation in reproductive traits within the population. These experiments tested only for variation in concentrations of these hormones in relation to reproduction. In fact,

Please cite this article as: White, J.T., et al., Insulins, leptin and feeding in a population of Peromyscus leucopus (white-footed mouse) with variable fertility, Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.02.006

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our results suggest that at least one of these hormones (leptin; Fig. 1c) is involved in reproductive modulation in P. leucopus. Conclusion Most importantly, we found no evidence supporting the hypothesis that heritable variation in reproductive phenotype might be due to heritable variation in circulating leptin, IGF-1, or insulin (Figs. 3, 5, 6). Where heritable variation exists in a system such as the reproductive axis, the cause of that variation might be (1) hormones and cells specifically within that system, or (2) the response to hormones, metabolic fuels, and other indicators of body condition or environment. Previously we found that the circadian system and photoperiodic time measurement (Majoy and Heideman, 2000), two systems with broad effects on physiology and behavior, do not cause reproductive variation between NR mice and R mice (Heideman and Pittman, 2009). Variation in levels of energetic hormones or metabolic fuels would also have multiple effects on many other systems (Schneider et al., 2013). This study suggests that blood glucose and three such hormones, leptin, IGF-1, and insulin, are apparently not an important cause of heritable variation in the reproductive axis in SD in this population. The converse may be true: that variation in the reproductive axis controls the activity of reproductive organs and reproductive behavior, which in turn may modify energy intake and metabolism. This could explain correlations between reproductive measures, food intake, and metabolism (Heideman et al., 2005; Kaseloo et al., 2012; Reilly et al., 2006). These findings may have relevance to natural populations of other mammals, including humans. Understanding the sources of genetic variation that affect phenotype and plasticity has potential ecological significance. Our results support the hypothesis that circulating hormones that affect multiple systems may be unlikely to cause normal intra-population heritable variation in reproductive life history traits. Instead, variation in reproductive life history traits may be due to variation in regulatory factors that are specific to the reproductive axis (Avigdor et al., 2005; Heideman et al., 2010; Kaseloo et al., 2012, in press). Acknowledgments We thank three anonymous reviewers for helpful comments and suggestions that improved the manuscript significantly. Thanks to P. Kaseloo, E. Bradley, P. Zwollo, M. Leu, J. Pittman, and H. Murphy for technical assistance. M. Crowell assisted with data collection, and L. Wright-Jackson with animal care. Recombinant leptin was provided by Amgen, Inc. Funding was provided by the National Institutes of Health (R15-HD068962), from a Howard Hughes Medical Institute Undergraduate Education Program Grant to the College of William and Mary, by the ALSAM Foundation, and the Charles Center of the College of William and Mary. References Adkins-Regan, E., 2008. Do hormonal control systems produce evolutionary inertia? Philos. Trans. R. Soc. Lond. B Biol. Sci. 363 (1497), 1599–1609. Afolayan, R.A., Fogarty, N.M., 2008. Genetic variation of plasma insulin-like growth factor1 in young crossbred ewes and its relationship with their maintenance feed intake at maturity and production traits. J. Anim. Sci. 86 (9), 2068–2075. Avigdor, M., Sullivan, S.D., Heideman, P.D., 2005. A response to selection for photoperiod responsiveness on the density and location of mature GnRH-releasing neurons. Am. J. Physiol. Regul. Integr. Comp. Physiol. 288, R1226–R1236. Barash, I.A., Cheung, C.C., Weigle, D.S., Ren, H., Kabigting, E.B., Kuijper, J.L., Clifton, D.K., Steiner, R.A., 1996. Leptin is a metabolic signal to the reproductive system. Endocrinology 137 (7), 3144–3147. Bartness, T.J., Wade, G.N., 1985. Photoperiodic control of seasonal body weight cycles in hamsters. Neurosci. Biobehav. Rev. 9 (4), 599–612. Bartness, T.J., Powers, J.B., Hastings, M.H., Bittman, E.L., Goldman, B.D., 1993. The timed infusion paradigm for melatonin delivery: what has it taught us about the melatonin signal, its reception, and the photoperiodic control of seasonal responses? J. Pineal Res. 15 (4), 161–190. Berrigan, D., Lavigne, J.A., Perkins, S.N., Nagy, T.R., Barrett, J.C., Hursting, S.D., 2005. Phenotypic effects of calorie restriction and insulin-like growth factor-1 treatment on body

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Please cite this article as: White, J.T., et al., Insulins, leptin and feeding in a population of Peromyscus leucopus (white-footed mouse) with variable fertility, Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.02.006