# Altered circadian pattern of activity in a chronic activity-based anorexia nervosa-like female mouse model deficient for GHSR

Julia Mattioni, Philibert Duriez, Sana Aïdat, Nicolas Lebrun, Mohammad Bohlooly-Y, Philip Gorwood, Odile Viltart, Virginie Tolle

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Julia Mattioni, Philibert Duriez, Sana Aïdat, Nicolas Lebrun, Mohammad Bohlooly-Y, et al. Altered circadian pattern of activity in a chronic activity-based anorexia nervosa-like female mouse model deficient for GHSR. Psychoneuroendocrinology, 2025, 177, pp.107453. 10.1016/j.psyneuen.2025.107453. hal-05177100

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# Altered circadian pattern of activity in a chronic activity-based anorexia nervosa-like female mouse model deficient for GHSR

Julia Mattioni<sup>a</sup>, Philibert Duriez<sup>a,b</sup>, Sana Aïdat<sup>a,d</sup>, Nicolas Lebrun<sup>a</sup>, Mohammad Bohlooly-Y<sup>c</sup>, Philip Gorwood<sup>a,b</sup>, Odile Viltart<sup>a,d</sup>, Virginie Tolle<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

- Anorexia Nervosa
- Circadian rhythms
- Ghrelin
- GHSR
- Physical activity

## ABSTRACT

Anorexia Nervosa (AN) is a psychiatric disorder characterized by voluntary food restriction, resulting in severe undernutrition that has been associated with circadian rhythms alterations. Yet, mechanisms that link circadian rhythm shifts to abnormal eating regulation in AN are poorly understood. Plasma ghrelin concentrations, an orexigenic hormone secreted by the stomach and acting through the GHSR (Growth Hormone Secretagogue Receptor), is elevated in restrictive-type AN. We aimed to test the hypothesis that GHSR signaling contributes to altered circadian pattern observed in AN. For this purpose, we first assessed whether chronotypes (morning, intermediate or evening) were different in patients with AN, bulimia nervosa and healthy controls. We next recorded the pattern of physical activity in young female GHSR deleted (GhrS-/-) and wild-type (GhrS+/-) mice housed in cages equipped with running wheels and exposed to quantitative food restriction, mimicking AN metabolic status. We demonstrated that chronotypes were different in the three groups of subjects, a difference mainly driven by an excess of morning chronotype in patients with AN. In mice, the shift toward higher daytime and pre-prandial physical activity and lower night-time and post-prandial physical activity, induced by food restriction, was impaired in GhrS-/- mice, suggesting a lack of capacity to adapt patterns of circadian activity to chronic food restriction. These data suggest an interaction between altered circadian pattern and AN and indicate that GHSR signaling deficiency may play a critical role in adapting circadian patterns of activity to the under-nutrition state in this disorder.

## 1. Introduction

Disruptions in circadian rhythms, which are 24-h rhythm controlled by both peripheral and central clocks, play a significant role in psychi- atric disorders, potentially contributing to their pathophysiology (Cretu et al., 2016; Allega et al., 2018; Wilcox et al., 2024). Food serves as an essential external cue for synchronizing circadian rhythms, and alter- ations in eating patterns commonly observed in eating disorders may lead to subsequent disruptions in these circadian rhythms, affecting activity levels, sleep-wake rhythms, social patterns or hormonal secre- tion, thereby exacerbating the disorders. Despite accumulating evidence of dysregulated 24-h rhythms in eating disorders, the precise mecha- nisms by which altered eating patterns impair circadian cycles remain

limited.

Anorexia nervosa (AN), known for its alarming mortality rate, the highest among all psychiatric disorders at 10 % per decade, is a severe eating disorder, primarily affecting women and typically emerging during adolescence and early adulthood (Duriez et al., 2023). It is characterized by altered patterns of food intake, marked by voluntary and persistent calorie restriction. Recent observational studies have linked AN to alterations in circadian rhythms related to hormonal secretion, sleep-wake cycle and rest-activity rhythm (Kandeğer et al., 2021; Menculini et al., 2019). Specifically, recent genome-wide associ- ation study (GWAS) identified a bidirectional genetic association be- tween AN and morning chronotype, a preference for higher activity and alertness during the early hours of the day, while insomnia has been

<footnote><sup>a</sup> Université Paris Cité, Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM U1266, Team "Vulnerability to Psychiatric and Addictive Disorders", Paris 75014, France</footnote>

<footnote><sup>b</sup> GHU-Paris Psychiatrie et Neurosciences, Hôpital Sainte Anne (CMME), F-75014 Paris, France</footnote>

<footnote><sup>c</sup> Translational Genomics, Discovery Sciences, Biopharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden</footnote>

<footnote><sup>d</sup> Université de Lille, SCALab - Sciences Cognitives et Sciences Affectives, CNRS UMR 9193, PsySEF Faculty, Villeneuve d'Ascq F-59653, France</footnote>

<footnote>* Correspondence to: Institute of Psychiatry and Neuroscience of Paris, 102-108 rue de la Santé, Paris 75014, France.
E-mail address: virginie.tolle@inserm.fr (V. Tolle).</footnote>
associated with a higher risk for AN, further highlighting the link between specific rest-activity rhythm and AN (Wilcox et al., 2024). These alterations in circadian rhythms not only manifest in rest-activity patterns but also in overall physical activity. Notably, AN is associated with impaired daily physical activity levels, with up to 80 % of individuals with AN engaging in excessive activity (Dalle Grave et al., 2008; Davis, 1997). These high levels of physical activity develop during the course of self-starvation and correlate with even poorer clinical outcomes (Dalle Grave et al., 2008; Davis, 1997; Epling et al., 1983). Numerous biological candidates may explain abnormal circadian rhythms in patients with AN, ghrelin being one of them.

Amongst peripheral sensors that help to adapt to the undernutrition state, ghrelin has been proposed to play a key role in the pathophysiology of AN. Ghrelin is a powerful orexigenic hormone synthesized and secreted by the stomach in anticipation of scheduled meals in both humans and rodents (Cummings et al., 2005; Verbaeys et al., 2011). Its effects are mediated through the Growth Hormone Secretagogue Receptor (GHSR), which is highly expressed, in key brain structures regulating appetite including the hypothalamus (Zigman et al., 2006). Individuals with AN but also rodents undergoing food restriction exhibit abnormally high plasma ghrelin levels, yet intriguingly, these elevated levels do not stimulate food intake in patients (Méquinion et al., 2015; Tolle et al., 2003). Interestingly, these high plasma ghrelin levels correlate with physical activity levels in AN individuals (Duriez et al., 2020), and with physical activity levels in anticipation of food, called food anticipatory activity (FAA), in mice (Tezenas du Montcel et al., 2023), while GHSR deleted mice demonstrate lower food anticipatory activity (LeSauter et al., 2009). Beyond that, energy balance interacts bidirectionally with the regulation of the circadian rhythm (Serin and Acar Tek, 2019), suggesting that changes in the circadian activity might contribute to the pathophysiology of AN. Taken together, these observations suggest a role of ghrelin signaling, during conditions of food restriction, in the circadian pattern of activity.

Food restriction and excessive physical activity characteristic of patients with AN have been replicated in rodents using the activity-based anorexia (ABA) model, which is widely used in research on AN. However, its effectiveness is limited by the short duration of the protocol, typically lasting only a few days. Given that AN is a chronic disorder, we previously developed and validated a modified version of the ABA protocol called the Food Restriction and Wheel (FRW) model (Méquinion et al., 2015). This chronic model involves quantitative food restriction coupled with free access to a running wheel, effectively mimicking key features of AN, including progressive weight loss, metabolic dysregulations, endocrine alterations such as disruption in estrous cycles, or alterations in circadian gene expression (Méquinion et al., 2015; Salaün et al., 2024). Yet, the neurobiological mechanisms underpinning the association between altered eating behaviors and disrupted circadian patterns of physical activity in AN remain largely unexplored.

The objective of this study was to investigate how GHSR deficiency affects the circadian pattern of physical activity in female mice submitted to chronic food restriction. More specifically, we aimed to test the hypothesis that ghrelin signaling contributes to altered circadian rhythm observed in patients with AN. For this purpose, we initially assessed whether morning *versus* intermediate/neutral or evening chronotype was different in a pilot study of patients suffering from eating disorders, either AN or Bulimia Nervosa (BN), and healthy controls. Furthermore, young female mice were exposed to quantitative food restriction in order to induce weight loss and elevation of plasma ghrelin, mimicking the physiological features of the disorder. Mice were housed in cages equipped with running wheels accessible at all times while the pattern of their physical activity was recorded.

## 2. Subjects and experimental procedures

### 2.1. *Patients and morningness-eveningness questionnaire*

Morningness and eveningness preference was tested with the Morningness-Eveningness Questionnaire (MEQ) (Horne and Ostberg, 1976). This test was administered to assess circadian typology, also defined as chronotype. The scale includes 19-items reflecting for example the preferred time for going to bed, getting up, the hour of the day when peak personal efficiency is at its maximum, the degree of tiredness within the first half hour after awakening and self-assessment of circadian type. The MEQ score is obtained by summing the scores for each item. Then, individuals are assigned to one of the three possible circadian typologies: < 41 evening-type; 42–58 intermediate/neutral-type; > 59 morning-type. We first checked if morning *versus* intermediate/neutral or evening chronotype was different in a pilot sample of patients with anorexia nervosa (AN, n = 22), compared to patients with bulimia nervosa (BN, n = 15) and healthy controls (HC, n = 9). Patients were recruited through a specialized eating disorder unit (Centre des Maladies Mentales et de l'Encéphale, CMME) in a University hospital department (Sainte-Anne hospital, Paris, France). The diagnosis of AN and BN patients were made during a face-to-face interview with a psychiatrist with at least five years of experience in diagnosis of eating disorders and according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association, 2022). Patients with AN, of either restrictive-type, i.e. presenting pure food deprivation (AN-R, n = 14) or binge-purgeing type, i.e. presenting both restrictive behavior and at least one bingeing/purging episode per week (AN-BP, n = 8) were included in the study. Healthy controls (BMI > 18.5 kg/m²) were recruited amongst members of the staff, mainly nurses, were all females, a past or current eating disorder being eliminated through the self-questionnaire SCOFF (Hill et al., 2010), a self-administered five-question test to assess the possible presence of an eating disorder. The committee for the protection of persons (CPP N° 2023-A02540-45) approved data collection and all subjects gave written consent.

### 2.2. *Animals and housing*

Experiments were performed on female GHSR knock-out mice (*Ghr*-/-; backcrossed on a C57BL/6 J genetic background). Mice were provided by AstraZeneca (GHSR knock-out mice originally obtained from Deltagen, San Carlos, CA, USA) (Egecioglu et al., 2010) and bred at the animal facility of the Institute of Psychiatry and Neuroscience of Paris (INSERM U1266, France). The colony was maintained from heterozygous breeding pairs (*Ghr*+/-) to obtain knock-out (*Ghr*-/-) and wild-type (*Ghr*+/-) littermates. At weaning, offspring were genotyped by ear notch PCR and mice were housed in a room under controlled illumination (6:00 am to 6:00 pm) and temperature (22-24°C). Mice had free access to chow diet (3 % fat, 16 % protein, 60 % carbohydrate, 4 % fibers, 2.79 kcal/g, Safe A04, Augy, France) and tap water unless otherwise stated. Experiments were conducted in a cohort of eight-week-old female *Ghr*-/- mice (n = 14) and *Ghr*+/- littermates (n = 8) at the beginning of the protocol. All experiments adhered to European Communities Council Directives (86/609/EEC) and received approval from the Regional Ethics Committee (CEEA.34) from Université Paris Cité.

### 2.3. *Food restriction and wheel protocol*

All mice were individually housed in Plexiglas cages, furnished with appropriate bedding, nesting materials, and an integrated running wheel (Actimetrics, Lafayette, IN, USA) placed in circadian cabinets with controlled 12-h light/dark light cycles (lights-off at 6:00 pm). Habituation to single housing and running wheel was performed over a 7-day period, during which mice had *ad libitum* access to food and
unrestricted access to the running wheel (ALW phase = *Ad libitum* + Wheel). Baseline 24-h food intake was monitored during this phase over a period of 4 days to determine 100% *ad libitum* food consumption. In the subsequent 16-day food restriction phase, mice were given 70% of their baseline food intake while maintaining unrestricted access to the running wheel (FRW phase = Food restriction + Wheel). Food pellets were provided daily at 05:00 pm. Daily monitoring of body weight (i.e., Day 0 of food restriction: 20.1 +/- 0.2 g in *Ghrsr+/-* and 19.9 +/- 0.1 g in *Ghrsr-/-*) allowed for adjustments in food delivery to ensure that body weight remained around 80% of initial body weight for each single animal (Fig. 1). This adjustment also aimed to ensure equal weight loss between groups, mitigating potential confounding effects of genotype and body weight on physical activity, given the strong correlation between body weight and diurnal physical activity (Supplementary Fig. 1S).

## 2.4. Running wheel activity monitoring

The running wheel activity, including the number of wheel revolutions and the duration of running, was recorded at 10-minutes intervals throughout the experimental protocol using MedPC software (Med-Associates, St Albans, VT, USA). Specifically, running activity was assessed daily during specific time intervals: 24 hours, light phase (06:00 am - 06:00 pm), dark phase (06:00 pm - 06:00 am), and the delta light (06:00 am- 06:00 pm) and dark (06:00 pmM - 06:00 am) periods. Additionally, running wheel activity was evaluated around mealtime: pre-prandial phase (from 03:00-05:00 pm), prandial phase (from 05:00-07:00 pm), post-prandial phase (from 07:00-09:00 pm) (Fig. 1).

## 2.5. Statistical analyses

Statistical analyses were performed using Prism version 10.2.0. For mouse data, assumptions of normality, homoscedasticity, independence of observations, and equidistance of factor levels were assessed to determine the appropriate statistical test. When these conditions were met, we used a two-way ANOVA with repeated measures to analyze the data; otherwise, a multiple Mann-Whitney test was utilized. For correlation analyses, for which normality was not verified, we used Spearman correlation. For clinical descriptive data, statistical differences were calculated with Kruskal-Wallis followed by post-hoc paired-wise

comparisons and differences in chronotype categorie using Chi-square test. Data are presented as mean ± SEM for animal data and as mean ± standard deviation for clinical data. For all statistical tests, significance was set at p < 0.05.

# 3. Results

## 3.1. Patients with AN have an excess of morning chronotype

We initially confirmed that morning/intermediate/evening chronotype had a trend for difference in the three groups (anorexia nervosa, bulimia nervosa and healthy controls) of subjects (Fisher $c^2$ = 6.09, df = 4, p = 0.05), a difference mainly driven by an excess of morning

<figure><img src="image_4.png" /></figure>

**Fig. 2.** Morningness and eveningness preference in patients with anorexia nervosa, bulimia nervosa and healthy controls. Morningness and eveningness preference was tested with the Morningness-Eveningness Questionnaire (MEQ) in patients with anorexia nervosa (AN, n = 22), bulimia nervosa (BN, n = 15) and healthy controls (CTL, n = 9). The proportion of individuals with a morning, neutral or evening chronotype was different in the three groups of subjects (p = 0.05), a difference mainly driven by an excess of morning chronotype in patients with AN compared to the rest of the sample (p = 0.009).

<figure><img src="image_5.png" /></figure>

**Fig. 1.** Experimental design of the FRW protocol. Mice were individually housed in homecages containing running wheels placed in circadian cabinets with controlled 12 h light/dark cycles (lights-off at 6:00 pm). Running wheels were connected to a software (MedPC, MedAssociates) that recorded the number of wheel revolutions at 10-minutes intervals and summed in order to obtain 12-h daytime and night-time activity as well as 24-h activity. Mice were given unrestricted access to the running wheel and *ad libitum* access to food for an initial period of 7 days (ALW = *Ad Libitum* and Wheel). Each mouse was then provided with 70% of its baseline food intake (measured in ALW conditions) during the following 16 days while maintaining unrestricted access to the running wheel (FRW = Food restriction and Wheel). Food pellets were provided daily at 05:00 pm.
chronotype (54.5 %) in patients with AN compared to the rest of the sample (16.7 %) ($c^2$ = 7.26, df = 1, p = 0.009) (Fig. 2).

BMI differed between patients with AN and healthy controls or patients with BN (p < 0.001) but not between BN and healthy controls. Eating Disorders Inventory (EDI-tot) and Hospital and Anxiety Depression Scales-Anxiety (HADS-A) and Depression dimensions (HADS-D) differed between healthy controls and AN (p < 0.001 for EDI-tot, p = 0.014 for HADS-A, p = 0.014 for HADS-A and p = 0.003 for HADS-B) and healthy controls and BN (p = 0.014 for HADS-A, p = 0.016 for HADS-A and p = 0.012 for HADS-B), but not between AN and BN (Table 1).

## 3.2. GHSR deleted mice have an impaired circadian pattern of running wheel activity during chronic food restriction

All mice received an equivalent percentage of food relative to their ad libitum baseline intake, with no significant differences in baseline food intake between groups (Fig. 3.A; Mann-Whitney test). Both Ghsr-/- and Ghsr+/+ mice maintained similar weights during both the ALW and FRW phases, with identical progressive weight loss during the FRW phase (Fig. 3.B; 2-group x 15-day ANOVA: p Genotype = 0.8793, p Day < 0.0001, p Days x Genotype = 0.0099). Both genotypes exhibited similar 24-hour levels of cumulative physical activity during the ALW and FRW phases (Fig. 3.C; 2-group x 22-day ANOVA: p Days x Genotype = 0.6670, p Days < 0.0001, p Genotype = 0.2886). Additionally, comparable levels of physical activity were observed during the dark phase in both groups during the ALW phase. However, Ghsr-/- mice exhibited a tendency for increased nocturnal activity during the FRW phase, particularly noticeable 9 days after the beginning of food restriction ("N9") (Fig. 3.D; 2-group x 22-day ANOVA; p Genotype = 0.0734, p Days x Genotype = 0.3581, p Days < 0.0001). Conversely, during the FRW light period, Ghsr-/- mice displayed reduced daily activity compared to Ghsr+/+ (Fig. 3.E; p Genotype = 0.0030, p Days x Genotype < 0.0001, p Days < 0.0001), while no difference was observed during the ALW phase. The difference of physical activity between night and day further revealed predominant activity during the nocturnal period in both genotypes during the ALW phase. However, during the FRW phase, Ghsr-/- mice maintained predominant activity during the night instead of transitioning to a majority of daytime activity (Fig. 3.F; p Genotype = 0.0035, p Days x Genotype < 0.0001, p Days < 0.0001), suggesting a failure to adjust their circadian pattern of physical activity while being submitted to chronic food restriction.

## 3.3. GHSR deleted mice have higher running wheel activity during the dark period and lower during the light period

Ghsr-/- and Ghsr+/+ mice exhibited similar average hourly patterns of physical activity during the ALW phase (Fig. 4.A; p Genotype = 0.7665; p Days < 0.0001; p Days x Genotype = 0.9912), with both genotypes showing significantly higher activity levels at night compared to the day (Fig. 4.C; p Genotype = 0.3392; p Food Restriction < 0.0001, p Genotype x Food Restriction = 0.4941). However, during the FRW phase, Ghsr-/- mice displayed a phase shift relative to Ghsr+/+ mice.

They exhibited higher hourly physical activity during the night and lower activity during the day, with a delayed onset and overall reduced levels of physical activity during the light phase compared to Ghsr+/+ (Fig. 4.B; p Genotype = 0.2874; p Days < 0.0001; p Days x Genotype < 0.0001). Similar to the ALW period, physical activity remained higher during the dark period compared to the light period in the FRW phase, with Ghsr-/- mice exhibiting higher activity levels at night and lower levels during the day (Fig. 4.D; p Genotype = 0.4098; p Food Restriction < 0.0001, p Genotype x Food Restriction = 0.0002).

## 3.4. GHSR deleted mice have an impaired pattern of physical activity around meal time

During the 2-hour period preceding food exposure, Ghsr-/- mice exhibited lower physical activity (Fig. 5.A; p Genotype = 0.0045, p Genotype x Time = 0.7725, p Time = 0.0324). Subsequently, during the 2-hour prandial phase, Ghsr-/- mice displayed reduced physical activity during the first 30 minutes after food return and resumed running approximately 50 minutes after food return, indicating a shift in activity levels (Fig. 5.B; p Genotype = 0.0227, p Time < 0.0001, p Genotype x Time < 0.0001). This shift led to Ghsr-/- mice ultimately exhibiting higher activity levels than Ghsr+/+ mice for the remainder of the prandial period, persisting throughout the 2-hour post-prandial period (Fig. 5.C; p Genotype = 0.0059, p Time < 0.0001, p Genotype x Time = 0.0007). These observed patterns before, during, and after the prandial phase were consistently reproduced throughout the protocol (Fig. 5.D; p Genotype = 0.0015, p Day < 0.0001, p Genotype x Day = 0.0498; Fig. 5.E; p Genotype = 0.3241, p Day < 0.0001, p Genotype x Time = 0.0497; Fig. 5.F; p Genotype = 0.0059, p Time < 0.0001, p Genotype x Time = 0.0480).

# 4. Discussion

This translational study aimed at better understanding the mechanism behind altered circadian rhythms in AN, a condition characterized by chronic undernutrition and elevated plasma ghrelin concentrations, and a possible link with defective GHSR signaling. After confirming that female patients with AN have abnormal circadian rhythms characterized by an excess of morning chronotype, our study emphasizes the critical role of GHSR signaling in modulating the circadian pattern of physical activity in female mice subjected to food restriction in a chronic ABA mouse model. We indeed observed that food restricted GHSR deleted mice display (i) lower physical activity in both the diurnal and pre-prandial period, (ii) higher physical activity in the post-prandial period, and (iii) lack of capacity to adapt patterns of circadian physical activity to chronic food restriction in order to favor daytime activity.

GWAS studies have recently re-conceptualized AN as a metabopsychiatric disorder (Watson et al., 2019), likely accompanied by disrupted circadian rhythms (Wilcox et al., 2024). Indeed, a recent mendelian randomization approach from Wilcox et al. (2024), using polygenic risk score for AN and sleep disorders, showed bidirectional associations between AN and morning chronotype, and between insomnia and higher risk for AN, suggesting that AN is yet another

**Table 1**

Clinical sample characteristics. Descriptive data are expressed with mean (Standard deviation). Statistical differences were calculated with Kruskal-Wallis followed by post-hoc pair-wise comparisons. BMI: Body Mass Index, EDI: Eating Disorder Inventory, HADS-A: Hospital Anxiety And Depression Scale (Anxiety dimension) and HADS-D Hospital Anxiety And Depression Scale (Depression dimension).

<table><thead><tr><th></th><th>Anorexia nervosa</th><th>Bulimia nervosa</th><th>Healthy control</th><th>Group Effect (p value)</th><th>AN vs BN</th><th>AN vs HC</th><th>BN vs HC</th></tr></thead><tbody><tr><td>n</td><td>22</td><td>15</td><td>9</td><td></td><td></td><td></td><td></td></tr><tr><td>Age (years)</td><td>25.5 (8.53)</td><td>29.3 (9.51)</td><td>28.4 (3.94)</td><td>0.045</td><td>0.124</td><td>0.110</td><td>0.661</td></tr><tr><td>BMI (kg/m<sup>2</sup>)</td><td>17 (2.66)</td><td>22.6 (3.32)</td><td>21.6 (1.53)</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>&lt; 0.001</td><td>0.969</td></tr><tr><td>Illness duration (months)</td><td>92.9 (95.8)</td><td>144 (94)</td><td>-</td><td>-</td><td>0.116</td><td>-</td><td>-</td></tr><tr><td>EDI-tot</td><td>115 (44.9)</td><td>124 (26.7)</td><td>18 (16.3)</td><td>&lt; 0.001</td><td>0.946</td><td>&lt; 0.001</td><td>&lt; 0.001</td></tr><tr><td>HADS-A</td><td>12.5 (3.36)</td><td>12.4 (3.25)</td><td>7.56 (4.13)</td><td>0.009</td><td>0.981</td><td>0.014</td><td>0.016</td></tr><tr><td>HADS-D</td><td>9.13 (4.73)</td><td>8.8 (4.62)</td><td>2.89 (3.26)</td><td>0.003</td><td>0.981</td><td>0.003</td><td>0.012</td></tr></tbody></table>
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** GHSR deleted mice have higher physical activity during the night and lower activity during the day in food restricted conditions. (A) Ghr~s~-/ and Ghr~s+/- mice received the same amount of food every day throughout the duration of the experiment. (B) Body weight loss during the FRW protocol was similar between Ghr~s~-/ and Ghr~s+/- mice. (C) Ghr~s~-/ mice exhibited similar daily amount of running wheel activity over 24 hours compared to Ghr~s+/- mice. (D) Ghr~s~-/ mice had a tendency toward higher daily night-time running wheel activity levels compared to Ghr~s+/- mice (p = 0.0734, RM Anova). (E) Ghr~s~-/ mice had significantly lower daily diurnal running wheel activity levels compared to Ghr~s+/- mice (p = 0.003, RM Anova). (F) The delta between daytime (6:00 am - 6:00 pm) and night-time (6:00 pm - 6:00 am) running wheel activity levels from D-6 to D16 shows that both genotypes had higher physical activity during the night in the ALW condition and that Ghr~s~-/ mice maintained significantly higher levels of physical activity during the night in the food restricted condition (p = 0.0035, RM Anova). Data are presented as mean ± SEM. ##p < 0.01: RM Anova, genotype effect; *p < 0.05, **p < 0.01, ***p < 0.001 Ghr~s~-/ vs Ghr~s+/-, post-hoc values for each time point. ALW = Ad Libitum and Wheel; FRW = Food Restriction and Wheel, RM= Repeated Measures.</figcaption></figure>

eating disorder with impaired circadian rhythms. An example of output of dysregulated circadian rhythms in AN is locomotor activity patterns, and, as previously mentioned, individuals with AN often exhibit elevated levels of physical activity (Duriez et al., 2021). Despite this, research on locomotor activity rhythms over a 24-hour period in AN remains scarce. While one study found increased activity around meal-time (Scheurink et al., 2010), our study is the first to offer a quantitative characterization of morning/intermediate/evening chronotype in the small sample tested herein. Yet, the role of abnormal eating rhythms in shaping altered circadian rhythms of physical activity, and the underlying biological mechanisms, both remain poorly understood, highlighting the importance of this study and the need for further investigations.

Ghrelin emerges as a key marker due to its association with AN, circadian rhythms, and the intricate interplay between the two. Ghrelin, a powerful orexigenic hormone primarily secreted by the stomach in response to energy deficit, is elevated in individuals with AN and following chronic food restriction in rodents (Hassouna et al., 2016). While peripheral ghrelin injection in rodents induces food intake (Barrile et al., 2023), the elevation of plasma ghrelin concentrations in individuals with AN does not lead to increased eating, suggesting impaired ghrelin signaling through GHSR and possibly ghrelin resistance for food intake in this disorder. Instead, elevated plasma ghrelin concentrations correlate with physical activity in both AN and undernourished mice (Duriez et al., 2020; Tezenas du Montcel et al., 2023). Interestingly, in mice with access to food during an 8-hour window in the light period, peripheral ghrelin injection induces an increase in physical activity during the period of food unavailability, thereby

altering the circadian rhythm for this activity (Sauter et al., 2009). However, it remains a question whether "variations in endogenous levels of ghrelin, such as through time-restricted feeding or fasting, are sufficient to produce changes in circadian rhythm" (Kulkarni et al., 2024). The fact that GHSR deleted mice maintain a higher night-time/daytime activity during calorie restriction may indicate a reduced ability to respond and adapt to changes in metabolic demands when food becomes less available. This is in contrast with previous studies showing improved metabolic flexibility in constitutive and neuronal-specific GHSR deleted mice (Longo et al., 2008; Lee et al., 2016), especially when switching from regular diet (RD) to high-fat (HF) diet, using carbohydrate as an energy source under RD while using fat as an energy source under HF diet conditions (Lee et al., 2016). The impact of GHSR deficiency in modulating metabolic flexibility in response to calorie restriction would need to be evaluated in our experimental conditions to draw conclusions (Rogers et al., 2016).

Our study sheds light on the role of GHSR signaling in shaping circadian rhythms of physical activity around mealtime. Specifically, we showed an adaptation to chronic calorie restriction characterized by increased pre-prandial activity, to facilitate access to food, a period associated with elevated plasma ghrelin concentrations, and a phase shift in daily physical activity/FAA characterized by an earlier onset of activity compared to mice with impaired GHSR signaling. These results are in part in accordance with the pioneer work of Le Sauter et al. (2009) who found reduced FAA and delayed onset of FAA in Ghr~s~-/ mice under a restricted feeding schedule with food access being limited to 8 hours per day with no weight loss in contrast to our model (Sauter et al., 2009). However, Le Sauter did not find an overall reduction in daily activity,
<figure><img src="image_7.png" /><figcaption>B 24h-Physical Activity (FRW)</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>C Total Physical Activitij (ALW)</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>D Total Physical Activity (FRW)</figcaption></figure>

<figure><img src="image_10.png" /></figure>

Fig. 4. Hourly distribution of running wheel activity shows impaired circadian pattern of physical activity in GHSR deleted mice in response to food restriction. (A) In *ad libitum* (ALW) conditions, *Ghr*-/- mice had a similar hourly pattern of running wheel activity than *Ghr*/+ mice (averaged over D-7 to D0). (B) In food-restricted (FRW) conditions, *Ghr*-/- mice showed an impaired hourly pattern of running wheel activity compared to *Ghr*/+ mice (averaged over D0 to D16), with higher activity during the night and lower activity during the day compared to *Ghr*/+ mice (p < 0.0001, RM Anova). *Ghr*-/- mice were the most active during the first 5 hours of the dark cycle while *Ghr*/+ mice were mostly active during the second half of the light cycle and the first half of the dark cycle. (C) In *ad libitum* (ALW) conditions, the relative quantity of daytime and night-time activity was similar in *Ghr*-/- and *Ghr*/+ mice. (D) In food restricted (FRW) conditions, the relative quantity of daytime and night-time activity was different in *Ghr*-/- and *Ghr*/+ mice (p = 0.0002, RM Anova). Data are presented as mean ± SEM. ###p < 0.01 and ####p < 0.0001 Night vs Day; \*p < 0.05, \*\*p < 0.01 *Ghr*-/- vs *Ghr*/+, post-hoc values. ALW = *Ad Libitum* and Wheel; FRW = Food Restriction and Wheel, RM = Repeated Measures. Gray box indicates night-time. Horizontal dark line in (B) indicates food anticipatory activity (FAA) period.

which might be attributable to different experimental conditions, with food given in the middle of the light period rather than at the end. Additionally, our findings question the conventional definition of FAA, typically characterized by increased physical activity occurring 4-6 hours before food return in the homecage (Scheurink et al., 2010). Indeed, in mice with normal GHSR signaling, we observed increased physical activity closed to the onset of the light period, starting to increase as early as 8 am, approximately 9 hours before food distribution. Other studies also support a role for ghrelin in regulating circadian activity or a disruption of the circadian activity in mice deficient for either ghrelin or the GHSR in a variety of experimental settings. Indeed, the ghrelin analog, GHRP-6 induces phase advances of locomotor activity after 30-h fasting, an effect that was not observed in *ad libitum* fed mice (Yannielli et al., 2007). In mice with limited access to food for 4 h daily in the middle of the light phase, anticipatory locomotor activity was attenuated by GHSR deletion (Blum et al., 2009). Lamont et al.

demonstrated that, under constant light exposure, locomotor activity of GHSR deleted mice was greater than WT mice in *ad libitum* access to food as well as in anticipation of the scheduled meal. Under constant darkness, GHSR deleted mice required more days to develop a high level of FAA (Lamont et al., 2014). Furthermore, deletion of GHSR amplifies the altered pattern of clock genes, such as Bmal1, Clock, and Per, in cultured hepatocytes isolated from obese mice (Wang et al., 2018). Nevertheless, most of those studies were performed in males (Wang et al., 2018) or the sex was not specified (LeSauter et al., 2009; Lamont et al., 2014; Blum et al., 2009). Considering that the ghrelin system is sexually dimorphic (Labarthe et al., 2022; Smith et al., 2022), our data performed in female mice in a chronic mouse model mimicking physiological features of AN have an important translational value and highlights the possible implication of GHSR signaling in a disorder that is mostly observed in young women. Interestingly, in healthy adults, circulating ghrelin levels are also regulated by the endogenous circadian cycle and by circadian
A

<figure><img src="image_11.png" /></figure>

B

<figure><img src="image_12.png" /></figure>

C

<figure><img src="image_13.png" /></figure>

D

<figure><img src="image_14.png" /></figure>

E

<figure><img src="image_15.png" /></figure>

F

<figure><img src="image_16.png" /><figcaption>**Fig. 5.** Peri-prandial running wheel activity in GHSR deleted mice during food restriction. (A) 2-h pre-prandial activity (averaged over D0-D16 and presented as 10-min intervals) was lower in *Ghr*-/- than in *Ghr*/+/+ mice (p = 0.0045, RM Anova). (B) 2-h prandial physical activity (averaged over D0-D16 and presented as 10-min intervals) was different in *Ghr*-/- and *Ghr*/+/+ mice with activity increasing with time in *Ghr*-/- but not *Ghr*/+/+ mice (p = 0.0227, RM Anova). (C) 2-h post-prandial running wheel activity (averaged over D0-D16 and presented as 10-min intervals) was higher in *Ghr*-/- than in *Ghr*/+/+ mice (p = 0.0059, RM Anova). (D) Sum of 2-h pre-prandial activity, measured daily from D0-D16, was reduced in *Ghr*-/- compared to *Ghr*/+/+ mice (p = 0.0015, RM Anova). (E) Sum of 2-h prandial activity, measured daily from D0-D16, was similar in *Ghr*-/- and *Ghr*/+/+ mice. (F) Sum of 2-h post-prandial activity, measured daily from D0-D16, was increased in *Ghr*-/- compared to *Ghr*/+/+ mice (p = 0.0059, RM Anova). Data are presented as mean ± SEM. T0 = Time of food distribution. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 *Ghr*-/- vs *Ghr*/+/+, post-hoc values for each time point. RM= Repeated Measures.</figcaption></figure>

misalignment in a sex-dependent manner (Qian et al., 2019). Overall, ghrelin signaling might be abnormal in AN since ghrelin blood concentrations are chronically elevated (Hassouna et al., 2016). One hypothesis is that under physiological conditions, ghrelin activates the reward system before a meal, thereby increasing the rewarding value of food (Egecioglu et al., 2010) and promoting physical activity to actively seek food. In AN, however, altered ghrelin signaling might disrupt these physical activity patterns. As high intensity exercise is associated with ghrelin suppression (Anderson et al., 2021), a vicious circle might be present where ghrelin facilitates the search for food through the adaptation of physical activity in intensity and diurnal patterns, while the observed physical activity would in turn impair ghrelin secretion. Such hypothesis suggests that ghrelin would indirectly contribute to altered circadian patterns in AN rather than directly influencing normal eating behaviors. We cannot definitely conclude that our observations in GHSR deleted mice are due to the absence of ghrelin action on the GHSR or are ghrelin-independent. Indeed, the lack of ghrelin signaling at the GHSR, the lack of GHSR constitutive activity (Fernandez et al., 2018) or the impaired ability of the GHSR to heterodimerize with other G-protein coupled receptors (Kern et al., 2012; Schellekens et al., 2013) could each contribute to the phenotype observed. Furthermore, plasma ghrelin concentrations were reduced acutely following food intake (data not shown), so differences observed during the post-prandial period may be ghrelin-independent. In the context of this translational study, measurement of plasma ghrelin concentrations to correlate with the patients chronotype would have been informative. However, circadian pattern of ghrelin to determine a time shift in the concentrations of the hormone in this context would have required a challenging exploration with blood samples withdrawn at regular intervals during the 24-h period (Germain et al., 2010).

Further studies should focus on the role of GHSR signaling on abnormal circadian rhythms of physical activity in AN through other circadian rhythms-related pathways. Such pathways could involve the suprachiasmatic nucleus (SCN), the body's master circadian regulator, and the lateral hypothalamus (LHA) including orexin (hypocretin) neurons, both of which play critical roles in circadian regulation and sleep/wake cycles, respectively (Ohno and Sakurai, 2008; Pandi-Perumal et al., 2022; Sanchez et al., 2022). The SCN is located in the hypothalamus and connect with structures implicated in the regulation of energy homeostasis, such as the hypothalamic arcuate nucleus that contains NPY/AgRP (Neuropeptide Y/Agouti-Related Pprotein) (Méndez-Hernández et al., 2020), which are direct targets of peripheral ghrelin and have been recently linked to encoding of circadian feeding activity (Cedernaes et al., 2019; Sayar-Atasoy et al., 2024). Bilateral electrolytic SCN lesioning experiments show alterations in symptoms related to AN, such as a modification in body weight, loss of circadian rhythms of oxygen consumption, and food intake, while exercising in a running wheel suppresses SCN neuronal activity in vivo (Hughes et al., 2021). Moreover, evidence suggests that ghrelin has direct actions on the SCN, since GHSR mRNA is expressed in the SCN, indicating a direct interaction between ghrelin and the circadian system (Zigman et al., 2006). Reciprocally, ghrelin secretion fluctuates in a circadian manner, suggesting a role for the SCN in regulating ghrelin secretion. This reciprocal interaction suggests that ghrelin and/or GHSR signaling may serve as a bridge between altered circadian rhythms of physical activity and abnormal metabolism in AN. However, the role of ghrelin and its receptor on the SCN are not yet thoroughly characterized, and further experiments are needed.

Another interesting system linking ghrelin to feeding and circadian activity is the orexin (hypocretin) system, which consists of two
neuropeptides secreted from orexin-containing neurons in the lateral hypothalamus. Orexin is involved in maintaining sleep and wakefulness; orexin neurons are activated during wakefulness, whereas during sleep they are inhibited (Sakurai, 2007). Early on publications have also highlighted the role of ghrelin in the regulation of sleep-wake cycle and the association between ghrelin secretion, feeding and sleep-wake pattern in both rodents (Tolle et al., 2002) and humans (García-García et al., 2014). More recent studies show that orexin neurons express the GHSR and are involved in ghrelin-induced orexigenic actions (Barrile et al., 2023). Furthermore, ghrelin and orexin interact to increase meal size (Suarez et al., 2020). In addition, orexin neurons also connect with the dopaminergic reward system in the ventral tegmental area (Lamont et al., 2012) and are involved in locomotor activity, which suggest important implications for AN pathophysiology. Finally, as recently demonstrated in mice, orexin neurons are activated during ABA and the FAA is strongly reduced when the dual orexin receptor antagonist, suvorexant, is injected intraperitoneally (Schéle et al., 2023). This aligns with the hypothesis that the primary evolutionary function of orexins is to promote foraging, whereby increased physical activity, enhancing the likelihood of finding food when it is scarce (Barson, 2020). However, whether orexins mediate physical activity in humans remains unclear, and the relevance of orexin signaling in AN requires further investigations.

## 5. Conclusion

Altogether, these data suggest a reciprocal interaction between altered circadian cycle and AN and indicate that GHSR signaling may play a role in regulating the circadian pattern of activity in response to food restriction in this disorder. Furthermore, considering that chronic disruption of the circadian cycle influences hunger, appetite and food preference (McHill et al., 2022), understanding mechanism linking undernutrition states and circadian activity may have important implications for therapeutic interventions in AN. Finally, although disruption in food availability can trigger alterations in metabolic hormones, such as ghrelin, and lead to many phenotypic traits of AN, AN remains a multifactorial disorder which etiology is largely unknown (Kaye, 2008). Food restriction should not be seen as a cause of the disorder but rather as one of the multiple risk factors that could lead to the development of AN in a specific context of other vulnerabilities.

### CRediT authorship contribution statement

**Tolle Virginie:** Writing – review & editing, Writing – original draft, Supervision, Resources, Funding acquisition, Conceptualization. **Viltart Odile:** Writing – review & editing, Project administration, Conceptualization. **Gorwood Philip:** Writing – review & editing, Resources, Funding acquisition. **Bohlooly-Y Mohammad:** Resources. **Lebrun Nicolas:** Formal analysis. **Aïdat Sana:** Methodology, Investigation. **Philibert Duriez:** Writing – review & editing, Visualization, Methodology, Formal analysis. **Mattioni Julia:** Writing – original draft, Methodology, Investigation, Formal analysis.

### Funding

Funding for this study was provided by Agence Nationale de la Recherche (ANR) grant ANR-19-CE37-0020-01 (to V.T.), Fondation de France (FdF) grant N°00081239-Remano (to P.G.) and Institut National de la Santé et de la Recherche Médicale (INSERM). ANR, FdF or INSERM had no further role in the study design, collection, analysis and interpretation of data, writing of the manuscript or decision to submit the article for publication.

### Declaration of Competing Interest

P. G. received during the last 5 years fees for presentations at

congresses or participation in scientific boards from Angelini, Biogen, Janssen, Lundbeck, Merk, Newron, Otsuka, Richter and Viatris. All other authors declare that they have no conflicts of interest.

### Acknowledgments

We would like to acknowledge all members of the animal facility at the Institute of Psychiatry and Neuroscience of Paris for the care of the animals and of the PhenoBrain Plateform (Ludivine Therreau and Gwenaëlle Le Pen) for providing support for mouse behavioral phenotyping. We warmly acknowledge Etienne Kimmel, Marie Fadigas, and the nurses from Clinique des Maladies Mentales et de l'Encephale (GHU Paris Psychiatrie et Neurosciences, Hopital Sainte-Anne, Paris, France) for the care of the patients and their contribution to the clinical study.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.psyneuen.2025.107453.

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