Title: β‐endorphin differentially contributes to food anticipatory activity in male and female mice undergoing activity‐based anorexia
Abstract: Physiological ReportsVolume 9, Issue 5 e14788 ORIGINAL ARTICLEOpen Access β-endorphin differentially contributes to food anticipatory activity in male and female mice undergoing activity-based anorexia Caitlin M. Daimon, orcid.org/0000-0003-2495-1372 Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, USASearch for more papers by this authorShane T. Hentges, Corresponding Author [email protected] Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, USA Correspondence Shane T. Hentges, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA. Email: [email protected] for more papers by this author Caitlin M. Daimon, orcid.org/0000-0003-2495-1372 Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, USASearch for more papers by this authorShane T. Hentges, Corresponding Author [email protected] Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, USA Correspondence Shane T. Hentges, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA. Email: [email protected] for more papers by this author First published: 04 March 2021 https://doi.org/10.14814/phy2.14788 Funding information: This work was funded by the National Institutes of Health (R01DK078749 to STH). AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Anorexia nervosa (AN) has a lifetime prevalence of up to 4% and a high mortality rate (~5–10%), yet little is known regarding the etiology of this disease. In an attempt to fill the gaps in knowledge, activity-based anorexia (ABA) in rodents has been a widely used model as it mimics several key features of AN including severely restricted food intake and excessive exercise. Using this model, a role for the hypothalamic proopiomelanocortin (POMC) system has been implicated in the development of ABA as Pomc mRNA is elevated in female rats undergoing the ABA paradigm. Since the Pomc gene product α-MSH potently inhibits food intake, it could be that elevated α-MSH might promote ABA. However, the α-MSH receptor antagonist SHU9119 does not protect against the development of ABA. Interestingly, it has also been shown that female mice lacking the mu opioid receptor (MOR), the primary receptor activated by the Pomc-gene-derived opioid β-endorphin, display blunted food anticipatory behavior (FAA), a key feature of ABA. Thus, we hypothesized that the elevation in Pomc mRNA observed during ABA may lead to increased β-endorphin concentrations and MOR activation to promote ABA. Further, given the known sex differences in AN and ABA, we hypothesized that MORs may contribute differentially in male and female mice. Using wild-type and MOR knockout mice of both sexes, a MOR antagonist and careful analysis of food anticipatory behavior and β-endorphin levels, we found 1) increased Pomc mRNA levels in both female and male mice that underwent ABA, 2) increased β-endorphin in female mice that underwent ABA, and 3) blunted FAA in both sexes in response to MOR genetic deletion yet blunted FAA only in males in response to MOR antagonism. The results presented provide support for both hypotheses and suggest that it may be the β-endorphin resulting from increased Pomc transcription that supports the development of some features of ABA. 1 INTRODUCTION Anorexia nervosa (AN) has a lifetime prevalence of up to 4% in women and less than 1% in men (Keski-Rahkonen & Mustelin, 2016; Smink et al., 2012) and has a high mortality rate at roughly 5–10% (Arcelus et al., 2011). Diagnostic criteria for AN include low bodyweight, intense fear of gaining weight, and disturbed body image perception (American Psychiatric Association, 2013). While not a formal criterion for diagnosis, excessive exercise is an extremely common feature observed in AN patients, with one study reporting the behavior in over 80% of those surveyed (Casper et al., 2020; Rizk et al., 2020). Unfortunately, no prevention or early intervention strategies for AN currently exist despite an obvious need. Moreover, while current treatment therapies are often initially effective, relapse frequently occurs (Khalsa et al., 2017). Despite the severity of this disease, surprisingly little is known regarding the neurobiological basis of AN (Zipfel et al., 2015). It is crucial that the gaps in knowledge be addressed to facilitate the development of novel therapeutics for AN. Studies using animal models have been instrumental in probing the underlying mechanisms of AN; one of the most widely used and well-established animal models is activity-based anorexia (ABA) (Klenotich & Dulawa, 2012). ABA closely mimics several key features of AN including severely restricted feeding and excessive exercise (Welch et al., 2018). In ABA, timed feedings restricted to limited hours of the day paired with access to a running wheel results in pronounced reductions in food intake and body weight loss as well as pronounced increases in wheel running activity. Wheel running activity is particularly increased in the hours preceding food presentation during the light cycle in which rodents are typically inactive, a phenomenon referred to food anticipatory activity (FAA; Mistlberger, 1994). Remarkably, animals will continue to lose weight and engage in wheel running to the point of exhaustion and death, as noted in the original reports describing this phenomenon over 50 years ago (Hall & Hanford, 1954; Routtenberg & Kuznesof, 1967). Sex differences have been reported in animals undergoing ABA though this difference remains incompletely understood as both sexes have been identified as more vulnerable to ABA compared to the other (females more vulnerable: Figure 1 in Klenotich & Dulawa, 2012, Pare et al. 1978; males more vulnerable: Achamrah et al. 2017; Doerries et al. 1991). Despite the evidence suggesting a sex difference is likely albeit incompletely understood, many ABA experiments previously where only conducted in one sex, usually female, given that females are diagnosed with AN in greater numbers than males (Zipfel et al., 2015). FIGURE 1Open in figure viewerPowerPoint Activity-based anorexia (ABA) can be reliably generated in wild-type mice. Wheel revolutions per 15-minute time bin over the course of 3 days in ABA are shown for female (a) and male (d) mice. Gray bars denote the dark cycle. Wheel revolutions during FAA, the four hours preceding food presentation, are shown in panels b (female, n = 8; *p = 0.0224) and E (male, n = 7; *p = 0.0334). Daily bodyweight expressed as a percentage of the animal's baseline average is shown in panels c and f for females and males, respectively. In both panels c and f, ****p < 0.0001. Summary data are presented as mean±SD. Data were analyzed using repeated measures one-way ANOVA. Additional details are found in the results section Using the ABA model, a role for the hypothalamic proopiomelanocortin (POMC) system has been implicated in the development of ABA as Pomc mRNA is transiently elevated in female rats undergoing ABA (Hillebrand et al., 2006). As a prohormone, POMC is enzymatically cleaved in multiple bioactive peptide products (Cawley et al., 2016; Harno et al., 2018) and previous investigations of POMC involvement in ABA have focused primarily on the cleavage product α-melanocyte stimulating hormone (α-MSH) given its ability to robustly inhibit feeding via activation of the melanocortin-4 receptor (Fan et al., 1997; Huszar et al., 1997). Yet while exogenous administration of α-MSH was found to exacerbate ABA (Hillebrand et al., 2005), subsequent experiments in which the melanocortin receptor antagonist SHU9119 was administered showed the drug incapable of ameliorating ABA (Hillebrand et al., 2006). Interestingly, it has also been shown that female mice lacking the mu opioid receptor (MOR), the primary receptor activated by the Pomc-gene-derived opioid β-endorphin, display blunted food anticipatory behavior, a key feature of ABA (Kas et al., 2004). In the current study, we hypothesized that the elevation in Pomc mRNA observed during ABA may lead to increased β-endorphin action and MOR activation to promote ABA. Further, given that AN disproportionately affects women, we hypothesized that MORs may contribute differentially in male and female mice. We first confirm the findings reported by Hillebrand and colleagues in female rats that Pomc mRNA is transiently elevated in female mice undergoing ABA as well as report a similar finding in male mice. We then show that circulating levels of β-endorphin increase in response to the ABA paradigm and that inhibiting MOR activation during ABA selectively reduces FAA but does not alter bodyweight or food intake in either male or female mice. Finally, we found a sex- and behavior-specific difference between the genetic deletion of MORs and pharmacologic inhibition of these receptors. These results indicate a potential sex-specific degree of involvement of the β-endorphin system in ABA and suggest that there could be a need for sex-specific approaches to treatment in patients with AN. 2 MATERIALS AND METHODS 2.1 Ethical approval All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals set forth by the National Institutes of Health and were approved by Colorado State University's Animal Care and Use Committee under protocol 19-9730A. All experiments comply with the ethical policies of the Journal of Physiology. 2.2 Animals Mice were initially acquired from the Jackson Laboratory (C57BL/6J, 000664 and B6.129S2-Oprm1tm1Kff/J, 007559) and bred at Colorado State University. Standard PCR techniques were used to genotype animals. Male and female mice aged 2–6 months were used in all experiments. Animals were maintained on a 12/12-hour light/dark cycle with ad libitum access to food and water unless stated otherwise. In the breeding room where animals were housed prior to entering an experiment, lights turned on at 06:00 hr. The experimental procedure room was on a modified light/dark cycle with lights turning on at 02:00 hr. Following transfer to the procedure room, all animals were given a minimum of 10 days to adjust to the altered light/dark cycle. During the acclimation period of ABA (discussed in detail below), wheel running activity was collected and analyzed to verify that proper adjustment to the altered light/dark cycle had occurred. All mice adjusted to the altered light/dark cycle prior to the start of the experiment. Room temperature was kept constant in both the breeding facility and the experimental test room at 20–22°C. 2.3 Activity-based anorexia model The activity-based anorexia (ABA) model is a well validated, commonly used behavioral paradigm in which access to a running wheel paired with restricted feeding results in severe weight loss and reductions in food intake, in addition to increased wheel running activity (Klenotich & Dulawa, 2012). At the start of all experiments, mice were singly housed in clean cages equipped with a running wheel (catalog # 0297; Columbus Instruments, Columbus, OH). Mice were given 3 days to acclimate to their new environment during which Multi Device Interface Software (Columbus Instruments, Columbus, OH) detected the total number of wheel revolutions every 15 minutes. Data were collected during acclimation for two reasons: first, to confirm proper adjustment to the altered light/dark cycle; and second, to determine whether the mouse exhibits sufficient baseline wheel running activity to warrant moving forward in the experiment. Mice running less than 1500 revolutions a day were considered nonrunners and were not used for ABA. Following the acclimation period, baseline daily bodyweight and food intake values were collected 1 hour prior to lights out for 5 days. Wheel running activity continued to be monitored every 15 minutes. Following baseline data collection, the ABA paradigm or relevant control condition was initiated. Food restricted animals were given access to chow for 2 hours a day, presented at the start of the dark cycle. Males and females were always run separately, and cages and wheels were thoroughly cleaned in between cohort runs. Control conditions included mice in cages where running wheels were provided but locked in place to create a food restricted without wheel running condition (FR only) and ad libitum fed animals provided access to a running wheel (WHL only). 2.4 Assessment of the temporal dynamics of Pomc mRNA expression or β-endorphin concentration For determination of Pomc mRNA and β-endorphin levels, male and female mice were sacrificed at lights out after varying lengths of exposure (1 day, 2 days, or 3 days) to the ABA paradigm (FR +WHL) or one of the control conditions. No differences in Pomc mRNA expression were detected in either control condition regardless of day of sacrifice; as such, control data were pooled. At sacrifice, animals were first deeply anesthetized with 200 mg/kg sodium pentobarbital solution (Fatal-Plus, Vortech Pharmaceuticals, Ltd, Dearborn, MI) and lack of deep pain reflex confirmed before transcardial perfusion with 10% sucrose followed with 4% paraformaldehyde in potassium phosphate-buffered saline. Brains were then stored at 4°C in 4% paraformaldehyde until sectioned. Whole blood was collected from the right atrium prior to perfusion and allowed to clot for 30 minutes at room temperature prior to centrifugation at 3000 rpm × 20 minutes at 4°C. Serum was removed and stored at −80°C. 2.5 Fluorescent in situ hybridization Fluorescent in situ hybridization was used to detect Pomc mRNA as previously described (Dennison et al., 2016; Jarvie et al., 2017). In brief, brains stored at 4°C in 4% paraformaldehyde were sliced into coronal sections (50 μM) spanning the rostral–caudal axis of the arcuate nucleus. Slices were then incubated at room temperature sequentially in: 6% hydrogen peroxide, Proteinase K (10 μg/ml), glycine (2 mg/ml), postfixation solution containing 4% paraformaldehyde, and 0.2% glutaraldehyde and finally ascending concentrations of ethanol prior to incubation in hybridization solution for 1 hr at 60°C (66% (v/v) deionized formamide, 13% (w/v) dextran sulfate, 60 mM NaCl, 1.3x Denhardt's solution, 13 mM Tris-HCL, pH 8.0, 1.3 mM EDTA, pH 8.0). The Pomc probe (0.25 pg/ml, corresponding to bases 531–1000 of GenBank sequence NM_08895.3) was denatured for 5 minutes at 85°C, added to the hybridization solution, and hybridized at 70°C for 18–20 hr. Brain slices were washed in saline sodium citrate buffer posthybridization before detection of the fluorescein isothiocyanate-labeled Pomc probe with a secondary antibody conjugated to Alexa Fluor 488 (1:400, Invitrogen/Thermo Fisher Scientific, Waltham, MA). Tissue sections were mounted on glass slides, cover-slipped, and stored at 4°C for later image collection and analysis. 2.6 Image collection and analysis All images were collected on a Zeiss 800 confocal microscope at 40x. Imaging parameters were kept consistent between experiments and each experiment contained both control and experimental animals. For each animal, a minimum of 10 tiled z-stack images taken from 10 brain slices at 1-μm intervals were obtained containing both sides of the arcuate nucleus. Pomc-expressing cells labeled with AlexaFluor-488 were identified using masks generated in ImageJ. The fluorescent intensity of each Pomc-expressing cell was expressed as a percentage of background fluorescence intensity for that given image. An overall average of fluorescent intensity above background was generated for each animal by averaging the values collected from individual z-stack images. 2.7 Radioimmunoassay Peptide extraction and β-endorphin measurement on previously stored serum samples were performed using a commercial radioimmunoassay kit according to the manufacturer's instructions (RK-022–33, Phoenix Pharmaceuticals, Inc., Burlingame, CA). In brief, samples were incubated overnight at 4°C with rabbit anti-β-endorphin antibody, followed by another overnight incubation with 125I-β-endorphin. Samples were then incubated with goat anti-rabbit IgG serum and normal rabbit serum, centrifuged, and the supernatant discarded prior to detection of bound 125I-β-endorphin in the remaining pellet with a gamma-counter (PerkinElmer, Waltham, MA). A standard curve was generated from which the concentration of β-endorphin present in each sample was extrapolated. The detection range of the kit used is 10–1280 pg/ml. 2.8 Disruption of MOR signaling To determine whether MORs contribute differentially in male and female mice to ABA, MOR function was inhibited in two ways: first by genetic deletion of the MOR using knockout mice discussed above, and second by administration of the MOR antagonist naloxone hydrochloride to wild-type animals (NAL, 5 mg/kg i.p., Sigma-Aldrich, St. Louis, MO). NAL was administered twice daily at 0.5 hours until lights out and again 4.5 hours later. Saline-treated control animals received two injections of 0.9% NaCl sterile saline solution (0.1 ml) at the same time. Animals were first habituated to i.p. injections of saline solution during baseline data collection (one injection per day). Unlike previous ABA experiments in which animals were sacrificed on a predetermined day, animals in these experiments were allowed to proceed through ABA uninterrupted until either 20% of initial bodyweight had been lost or 6 days had passed, at which point all animals were removed from the study. Upon completion of the experiment, animals were humanely euthanized. 2.9 Statistical analyses Detailed information regarding specific statistical tests used is given in the results section. All data were analyzed using Prism (GraphPad Software Inc., San Diego, CA). Data are presented as mean ± SD. Differences were considered significant when p ≤ 0.05. 3 RESULTS 3.1 Activity-based anorexia (ABA) can be reliably generated in wild-type mice One of the most widely used and well-established animal models is activity-based anorexia (ABA) as it closely mimics several key features of AN, including severely restricted feeding and excessive exercise (Klenotich & Dulawa, 2012). We first verified that we were able to reproduce the increased wheel running during the hours preceding food presentation known as food anticipatory activity (FAA) as well as bodyweight loss in response to restricted feeding in female and male wild-type mice (Figure 1). Data from female mice are presented in panels a-c. Data from male mice are presented in panels d-f. Wheel revolutions per 15-minute bin over the course of 3 days in ABA are first shown for either sex (Figure 1a,d). The daily cumulative total of wheel revolutions run during FAA, the four hours preceding food presentation, are shown in panels b (female) and e (male). As an ABA experiment progresses, the sample size inevitably gets smaller given that animals are removed from an ABA study when they lose 20% of their baseline bodyweight or greater. We have therefore elected to display the data up to the point that removal of animals from the experiment became necessary. For females, this is up to 4 days of ABA; for males, 3 days. In both sexes, we were able to reliably observe a significant increase in FAA in response to the ABA paradigm. In females, repeated measures one-way ANOVA revealed a significant overall effect after 4 days of ABA: F(1.784, 12.49) = 5.45, p = 0.0224 (Figure 1b, n = 8, 24 hr: 29 ± 40.48 mean ± SD, 48 h: 402.6 ± 709.1, 72 hr:2615 ± 3481, 96 hr: 5085 ± 4907). Repeated measures one-way ANOVA revealed a significant overall effect in males after 3 days of ABA: F(1.129, 5.646) = 7.575, p = 0.0334, n = 6, 24 hr: 202.3 ± 403.8, 48 hr: 1915 ± 2352, 72 hr: 6099 ± 5343 (Figure 1e). Bodyweight loss over the course of ABA is shown as the animal's daily bodyweight expressed as a percentage of its baseline average for both females (Figure 1c; repeated measures one-way ANOVA; F(1.309, 9.164) = 71.35, p < 0.0001, n = 8, 24 hr: 93.12%±1.954, 48 hr: 88.61%±1.494, 72 hr: 84.63% ± 2.474, 96 hr: 82.19%±3.315) and males (Figure 1f; repeated measures one-way ANOVA; F(1.559, 10.92) = 77.61, p < 0.0001, n = 8, 24 hr: 92.31% ± 2.694, 48 hr: 86.72% ± 3.278, 72 hr: 82.70% ± 3.464). Relative to the night before the initiation of ABA, a significant decrease in food intake was observed when hours of food access were restricted to the first two hours of the dark cycle in both females and males (Table 1). TABLE 1. Food intake data expressed as a percentage of the animal's baseline average food consumption Sex Condition Food consumed as percentage of baseline average (mean ± SD) 0 hr in ABA 24 hr in ABA 48 hr in ABA 72 hr in ABA 96 hr in ABA Female WTa n = 8, 99.26%±15.31 n = 8, 23.43%±8.799 n = 8, 38.75%±7.506 n = 8, 40.94%±9.255 n = 8, 45.30%±6.763 Female MOR kob – n = 9, 15.48%±4.751 n = 9, 29.35%±8.339 n = 9, 32.88%±7.735 n = 9, 41.86%±10.35 Male WTc n = 7, 110%±4.793 n = 7, 17.83%±9.297 n = 8, 46.67%±13.26 n = 8, 39.34%±11.12 – Male MOR kod – n = 13, 13.10%±3.653 n = 13, 33.99%±11.66 n = 13, 31.25%±11.72 – Male NALc – n = 9, 18.70%±4.780 n = 8, 41.98%±10.69 n = 8, 43.49%±10.89 – All data presented as mean ± SD. a) One-way repeated measures ANOVA in WT females p < 0.001; F(2.125, 14.88) =94.12. b) Two-way repeated measures ANOVA comparing MOR knockout females to WT females followed post-hoc analysis via Sidak's multiple comparisons. Sidak's multiple comparisons were not significant (24 hr WT vs. MOR ko:p = 0.1679; 48 hr WT vs. MOR ko: p = 0.1047, 72 hr WT vs. MOR ko: p = 0.2625; 96 hr WT vs. MOR ko: p = 0.8916) despite an overall effect (p = 0.149; F(1,15) =7.565). c) One-way repeated measures mixed effects model to account for missing datapoint due to data collection error in WT males; p < 0.001; F(3,26) =105.5. d) Two-way repeated measures mixed effects model comparing MOR knockout males to WT males followed by post-hoc analysis via Sidak's multiple comparisons. Sidak's multiple comparisons were not significant (24 hr WT vs. MOR ko:p = 0.555; 48 hr WT vs. MOR ko: p = 0.1256, 72 hr WT vs. MOR ko: p = 0.3481) despite an overall effect (p = 0.003; F(1,56) =9.647). e) Two-way repeated measures mixed effects model; p = 0.9688; F(1,42) =0.001543. 3.2 Pomc mRNA is transiently increased in both female and male animals undergoing ABA Pomc mRNA levels change in response to an organism's energy state such that during times of positive energy balance Pomc mRNA is increased (Schwartz et al., 1997) and during times of negative energy balance Pomc mRNA is decreased (Benoit et al., 2002; Mizuno et al. 1998). Paradoxically, Hillebrand and colleagues have previously shown a transient increase in Pomc mRNA levels in female rats undergoing ABA despite the animal existing in a state of negative energy balance (Hillebrand et al., 2006). We performed fluorescent in situ hybridization to detect changes in Pomc mRNA levels in female and male wild-type mice having undergone one, two, or three days of ABA or a control condition (food restriction only or wheel running only). Example images are shown in Figure 2a. The fluorescent intensity of each Pomc-expressing cell was expressed as a percentage of background fluorescence intensity specific to that image and an overall average of fluorescent intensity above background was determined for each animal for statistical analysis. One-way ANOVA revealed a significant difference in means between treatment groups (Figure 2b, F(4,21) = 3.892, p = 0.0162). After one day of ABA, female mice showed a significant increase in Pomc mRNA fluorescent intensity compared to both food restricted controls (Figure 2b, Tukey's multiple comparison, *p = 0.0500, FR only: n = 6, 223.2%±29.75, 24 h ABA: n = 5, 322.9%±78.83) and wheel running only controls (Figure 2b, Tukey's multiple comparison, #p = 0.0177, WHL only: n = 6, 207.1%±71.95, 24 hr ABA: n = 5, 322.9%±78.83). Female mice showed a peak in fluorescent intensity after one day of ABA. By thee days of ABA, fluorescent intensity values had essentially returned to levels observed in either control condition (Figure 2b, Tukey's multiple comparison, FR only vs. 72 hr ABA: p = 0.9997, FR only: n = 6, 223.2% ± 29.75, 72 hr ABA: n = 3, 216.2% ± 42.06, WHL only vs. 72 hr ABA: p = 0.9993, WHL only: n = 6, 207.1% ± 71.95, 72 hr ABA: n = 3, 216.2% ± 42.06). FIGURE 2Open in figure viewerPowerPoint Pomc mRNA is transiently elevated in both female and male mice undergoing ABA. Representative confocal images taken at 40× of Pomc mRNA detected with Alexa488-labeled probe in the arcuate nucleus of the hypothalamus from male animals sacrificed after 48 hr (ai) or 72 hours (aii) in ABA. Summary data from females are shown in panel b: *p = 0.0500, #p = 0.0177, one-way ANOVA. Summary data from males are shown in panel c: *p = 0.0110, one-way ANOVA. Summary data are presented as mean ± SD. Additional details are found in the results section. 3 V = third ventricle. FR = food restricted. WHL = wheel. n numbers were as follows: female: FR only: n = 6, WHL only: n = 6, 24 hr ABA: n = 5, 48 hr ABA: n = 6, 72 hr ABA: n = 3, male: FR only: n = 4, WHL only: n = 5, 24 hr ABA: n = 5, 48 hr ABA: n = 5, 72 hr ABA: n = 6 As observed in the female mice, a significant difference in mean fluorescent intensity was observed between treatment groups in male mice (Figure 2c, one-way ANOVA, F(4,19) =4.398, *p = 0.0110). Unlike in females where the peak fluorescent intensity was observed after one day of ABA (Figure 2b, 24 hr ABA: n = 5, 322.9%±78.83), in male animals the peak fluorescent intensity was observed after two days of ABA (Figure 2c, 48 hr ABA: n = 5, 291.9%±32.71). Fluorescent intensity was significantly decreased by three days of ABA compared to two days of ABA (Figure 2c, Tukey's multiple comparison, 72 hr ABA vs. 48 hr ABA: **p = 0.0098, 72 h ABA: n = 6, 179.6%±17.66, 48 h ABA: n = 5, 291.9% ± 32.71). No significant difference was observed between either control condition and thee days of ABA (Figure 2c, Tukey's multiple comparison, FR only vs. 72 hr ABA: p = 0.9756, FR only: n = 4, 198.1 ± 75.10, 72 hr ABA: n = 6, 179.6%±17.66, WHL only vs. 72 hr ABA: p = 0.9130, WHL only: n = 5, 204.7%±39.34, 72 hr ABA: n = 6, 179.6%±17.66). 3.3 Peripheral levels of β-endorphin increase over the course of ABA To determine whether the increase in Pomc mRNA observed might lead to an increase in circulating levels of β-endorphin, we performed radioimmunoassays to detect levels of β-endorphin in serum from animals euthanized after varying days in ABA. One-way ANOVA revealed a statistically significant effect in female mice (Figure 3a, F(2,14) =4.701, p = 0.0274). Specifically, a significant increase in β-endorphin was observed when female mice completed 3 days of ABA compared to 1 day (Figure 3a, Tukey's multiple comparison, p = 0.0223, 24 hr ABA: n = 5, 71.25 ± 12.82, 72 hr ABA: n = 7, 176.2 ± 81.78). No significant difference was found between one and two days in ABA (Figure 3a, Tukey's multiple comparison, p = 0.3940, 24 hr ABA: n = 5, 71.25 ± 12.82, 48 hr ABA: n = 5, 121.4 ± 43.79) or two and three days in ABA (Figure 3a, Tukey's multiple comparison, p = 0.2817, 48 hr ABA: n = 5, 121.4 ± 43.79, 72 hr ABA: n = 7, 176.2 ± 81.78). Mean concentration of β-endorphin from male mice is presented in Figure 3b, though statistical analysis was not performed due to the low number of samples from male mice that met quality standards (Figure 3b, 24 hr ABA: n = 3, 73.18 ± 16.92, 48 hr ABA: n = 3, 86.87 ± 30.99, 72 hr ABA: n = 4, 92.08 ± 21.24). FIGURE 3Open in figure viewerPowerPoint Peripheral levels of β-endorphin are elevated in response to ABA. Radioimmunoassay was used to detect circulating levels of beta-endorphin in peripheral blood collected from female (a) and male (b) after varying days of exposure to ABA. Data are presented as mean ± SD. *p = 0.0223. Data were analyzed using one-way ANOVA. Additional details are found in the results section. n numbers were as follows: female: 24 hr ABA: n = 5, 48 hr ABA: n = 5, 72 hr ABA: n = 7, male: 24 hr ABA: n = 3, 48 h ABA: n = 3, 72 hr ABA: n = 4 3.4 Food anticipatory activity (FAA) is blunted after MOR deletion in both male and female mice After detecting elevations in Pomc mRNA and β-endorphin concentration in response to ABA, we next investigated whether MOR activation contributes to the development of ABA. Previous studies have shown that FAA can be blunted in female MOR knockout mice undergoing ABA (Kas et al., 2004); given the known sex differences in AN and ABA however, we hypothesized that MOR activation may contribute differentially to the development of ABA in males versus females. Data from female and male MOR knockout mice are shown in Figure 4. The overall pattern of wheel revolutions per 15-minute bin over the course of 3 days in ABA is shown for female (Figure 4a) and male (Figure 4d) mice (wild-type animals in black, MOR knockout animals in blue). There is day-to-day variability in FAA for any given animal and a distribution in days needed to lose 20% of initial bodyweight, and thus, FAA is presented for each animal at the day that wheel revolutions were highest for the individual. In the majority of instances, the highest lev