Skip to main content
Download PDF

Sex-dependent circadian alterations of both central and peripheral clock genes expression and gut–microbiota composition during activity-based anorexia in mice

Biology of Sex Differences volume 15, Article number: 6 (2024) Cite this article

Abstract

Rationale

Patients with anorexia nervosa (AN) often present sleep disorders and circadian hormonal dysregulation. The role of the microbiota–gut–brain axis in the regulation of feeding behavior has emerged during the last decades but its relationships with the circadian rhythm remains poorly documented. Thus, we aimed to characterize the circadian clock genes expression in peripheral and central tissues in the activity-based anorexia mouse model (ABA), as well as the dynamics of the gut–microbiota composition.

Methods

From day 1 to day 17, male and female C57Bl/6 mice were submitted or not to the ABA protocol (ABA and control (CT) groups), which combines a progressive limited access to food and a free access to a running wheel. At day 17, fasted CT and ABA mice were euthanized after either resting (EoR) or activity (EoA) phase (n = 10–12 per group). Circadian clock genes expression was assessed by RT-qPCR on peripheral (liver, colon and ileum) and central (hypothalamic suprachiasmatic nucleus or SCN) tissues. Cecal bacterial taxa abundances were evaluated by qPCR. Data were compared by two-way ANOVA followed by post-tests.

Results

ABA mice exhibited a lower food intake, a body weight loss and an increase of diurnal physical activity that differ according with the sex. Interestingly, in the SCN, only ABA female mice exhibited altered circadian clock genes expression (Bmal1, Per1, Per2, Cry1, Cry2). In the intestinal tract, modification of clock genes expression was also more marked in females compared to males. For instance, in the ileum, female mice showed alteration of Bmal1, Clock, Per1, Per2, Cry1, Cry2 and Rev-erbα mRNA levels, while only Per2 and Cry1 mRNAs were affected by ABA model in males. By contrast, in the liver, clock genes expression was more markedly affected in males compared to females in response to ABA. Finally, circadian variations of gut–bacteria abundances were observed in both male and female mice and sex-dependent alteration were observed in response to the ABA model.

Conclusions

This study shows that alteration of circadian clock genes expression at both peripheral and central levels occurs in response to the ABA model. In addition, our data underline that circadian variations of the gut–microbiota composition are sex-dependent.

Plain language summary

Anorexia nervosa is an eating disorder with a female predominance. However, the underlying pathophysiological mechanisms are still incompletely understood. Patients with anorexia nervosa often show alterations in circadian rhythm, including sleep disorders and modifications in hormone circadian rhythm. The circadian rhythm is controlled in the central nervous system, particularly in the suprachiasmatic nucleus, but clocks have also been described in peripheral tissues. To better understand the putative role of circadian rhythm in the pathophysiology of anorexia nervosa, we have conducted an experimental study in a rodent model of anorexia nervosa called “activity-based anorexia” on both males and females. Interestingly, we observed that the expression of genes involved in the circadian rhythm is affected by the activity-based anorexia model in both the suprachiasmatic nucleus and peripheral tissues, such as the small intestine and liver. In addition, gut–microbiota also shows circadian variation. Interestingly, the anorexia-induced alterations of circadian variations (clock genes expression and gut–microbiota composition) are sex- and tissue-dependent. For instance, female mice exhibited more marked alterations in the ileum, whereas, in males, modifications were more pronounced in the liver. This study highlights sex-dependent alterations of circadian clock genes expression and of gut–microbiota in response to the anorexia rodent model. Further experiments should be performed to investigate the contribution of these mechanisms in the etiology of anorexia nervosa and the higher prevalence in females.

Highlights

  • In response to activity-based anorexia, a rodent model mimicking anorexia nervosa, the activity wheel is affected in a sex-dependent manner.

  • In response to activity-based anorexia, circadian clock genes changes are sex- and tissue-dependent. In peripheral tissues, males and females showed more marked alterations of circadian clock genes expression, respectively, in the liver and in the ileum.

  • Gut–microbiota composition is differently affected by activity-based anorexia in female and male mice.

Introduction

Anorexia nervosa (AN), an eating disorder defined by the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) [9], is characterized by a body mass index (BMI) lower than 18.5 kg/m2, an intense fear of gaining weight leading to a negative energy balance. AN is also frequently associated with compensatory mechanisms, such as physical hyperactivity in 30–80% of patients [14, 18] and an altered body shape perception. In addition, AN is the psychiatric disorder with the highest mortality rate, around 1–5% after 10 years [27]. The lifetime prevalence of AN is increasing with about 1.4% in women and 0.2% in men [22]. Nevertheless, studies in both females and males are needed because of differences in the pathophysiology, in the incidence peaks, in brain development and hormonal changes [48]. The etiology of AN is multifactorial and remains not well-understood [57]. During the last decades, alterations of metabolic pathways and the role of microbiota–gut–brain axis in AN have been highlighted. Indeed, a gut–microbiota dysbiosis has been reported in several studies both in AN patients [5, 6, 20] and in animal models [6, 7, 17, 51, 52]. In addition, sleep disorders [2, 3] and dysregulations of hormonal circadian rhythm [26] have been reported in AN patients. For instance, circadian plasma leptin levels are altered in AN patients and are associated with insulin and cortisol levels [26].

Circadian rhythm is regulated by a master clock in the suprachiasmatic nucleus (SCN) that is mainly controlled by light/dark cycle, while peripheral clocks are impacted by food intake. The primary actors involve CLOCK/BMAL1 (also named ARNTL) couple interaction regulated by PER/CRY couple. At the second level of regulation, NR1D1 (commonly named REV-ERBΑ) regulates Bmal1 transcription. Clock genes expression differs between tissues [30, 55] as referenced in the database of circadian transcriptional profiles, CircaDB [39]. Interestingly, gut–microbiota also seems to affect peripheral clocks, particularly in the intestine [45, 47]. Germ-free mice exhibit changes in liver circadian genes expression, such as Cry1, Per1 and Per2 [34]. By contrast, disruption of circadian rhythm in mice induced modifications: of the intestinal barrier function, of bacteria recognition pathways and of gut–microbiota composition with decreased Lactobacillus johnsonnii abundance [16]. These data underline that circadian rhythm alterations might affect the microbiota–gut–brain axis and might contribute to AN pathophysiology.

The most relevant rodent model for AN is the activity-based anorexia (ABA) model [43, 44]. Developed in both rats and mice, it consists in a reduction of the duration to food access combined with a free access to a running wheel to mimic both the undernutrition and the hyperactivity observed in AN patients. ABA rodents showed a reduced food intake, a marked body weight loss [29, 37], a dysbiosis of the gut–microbiota [7, 52], reduced brain volumes [52], a decrease of leptin plasma level [49], as well as circadian rhythm alterations with a diurnal food anticipatory activity or FAA [1, 21]. In addition, a sex-dependent response to ABA was previously described. Males showed more marked responses to the ABA model with a lower food intake and a higher body weight loss than females, associated with sex-dependent alterations of physical activity and food behavior regulatory neuropeptides levels [1, 50]. In addition, the response to drugs or interventions also appears sex-dependents, such as the response to ketamine injections [24], to naloxone treatment [13] or to gut–microbiota depletion [49].

Thus, we aimed to characterize the circadian transcriptional profile at the central and peripheral levels in both female and male mice submitted to the ABA model. In addition, we also evaluated the circadian changes in gut–microbiota composition in this animal model of anorexia.

Materials and methods

Experimental procedure

All experimental procedures were approved by an ethical committee (APAFIS#9473-2016072016008305 v6). After 6 days of acclimatization to inversed cycle (dark phase between 10:00 AM and 10:00 PM) and a standard diet (SAFE A03 SP-10, Augy, France) in the SCAC animal behavioural platform, 48 male and 48 female 9-week-old C57BL/6 mice (Janvier Labs, Le Genest-Saint-Isle, France) were randomly assigned to either the control (CT) or activity-based anorexia (ABA) groups. CT mice were placed in standard cages and ABA mice were housed in cages equipped with a monitored running wheel (ActiviWheel software Intellibio, Seichamps, France). CT mice had free access to food and water throughout the study. After 5 days of acclimatization with free access to food and water, ABA mice had a limited access to food from 6 h/day at day 6 to 3 h/day at day 9 and until the end of experiment at day 17. Food access was provided at the beginning of the dark phase (10:00 AM). Food intake and body weight were registered daily at the end of the light phase (Additional file 1: Fig. S1). For ethical reasons, two males ABA mice were excluded from the protocol.

To investigate a putative shift in circadian rhythm, mice were euthanized at two timepoints: at the end of the activity phase (EoA), between 9:00 PM and 11:00 PM, or at the end of the resting phase (EoR), between 9:00 AM and 11:00 AM. Thus, there were four groups for both females and males: CT–EoA/CT–EoR/ABA–EoA/ABA–EoR with n = 11–12 per group (Additional file 2: Table S1). To avoid interaction of food intake on circadian clock genes expression, pellets were removed from both CT and ABA mice at 1:00 PM. After 9 or 21 h of fasting for EoA or EoR groups, respectively, mice were anesthetized with an intraperitoneal injection of Ketamine (Boehringer Ingelheim, COVETO, Caen, France; 100 mg/kg) and Xylazine (Bayer HealthCare, Puteaux, France; 10 mg/kg) diluted in NaCl 0.9%. Then, blood was collected on heparinized vacutainer tubes in the abdominal aorta and tissues were sampled as described in the Additional file 1 (Fig. S1). Blood samples were then centrifuged at 3000×g for 15 min at 4 °C and plasma was collected. All samples (plasma, hypothalamus containing SCN, liver, colon, ileum, and cecal content) were immediately frozen in liquid nitrogen and stored at − 80 °C until analysis (see Additional file 1: Fig. S1 for details).

Table 1 Primers used to study circadian rhythm by RT-qPCR
Full size table

RT-qPCR

TRIzol-chloroform (Invitrogen, Carlsbad, CA, USA and Merck, Darmstadt, Germany, respectively) RNA extraction was performed as previously described [10, 12] on SCN, liver, colon and ileum. After DNAse treatment (Promega, Charbonnières-les-Bains, France), reverse transcription of RNAs into cDNAs was performed with M-MLV (Invitrogen, Carlsbad, US). The circadian genes studied were Rev-erbα, Bmal1, Clock, Per1 and 2, Cry 1 and 2 and housekeeping gene was Gapdh. Primers used for circadian rhythm are displayed in Table 1. All qPCRs used SYBR Green technology (Bio-Rad Laboratories, Marnes la Coquette, France) and were performed on an Eppendorf RealPlex Mastercycler (Eppendorf, Hambourg, Germany) or on a Bio-Rad CFX96 manager (Bio-Rad Laboratories, Marnes la Coquette, France). The values were obtained by the conversion of cycle threshold on concentration value using a standard curve. mRNA levels of genes of interest were normalized by mRNA levels of Gapdh.

Gut–microbiota analyses

DNA was extracted from cecal content and qPCR were performed as previously described [7], using QIAmp DNA stool kit (Qiagen, Hilden, Germany). Then, qPCRs were performed on Eppendorf RealPlex Mastercycler or on a Bio-Rad CFX96 manager to determine the abundance of different bacterial taxa. The taxa-specific primers used for gut–microbiota quantification are displayed in Table 2.

Table 2 Primers used for gut–microbiota abundance on cecal DNA with qPCR
Full size table

Statistical analyses

Statistical analyses and graphs were performed with GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA). Data of body weight, food intake and activity were analyzed with two-way ANOVA (Sex x Time) with Holm–Sidak's or Dunnett’s multiple comparison test. T test were used to compared specifically two points. Outliers from qPCR data were identified with Rout 1% statistical test and excluded. qPCR data were then analyzed with two-way ANOVA (ABA x Euthanized time) with Tukey post hoc tests. Data are expressed as mean ± standard error to mean. Concerning all statistical tests, p < 0.05 was considered as significant results. In each qPCR graph, significant two-way ANOVA p values for the ABA model (ABA), the circadian rhythm (CR) or for interaction (Int) are shown in bold and underlined text (ABA, CR or Int). Tukey post-tests p values were mentioned with symbols when p < 0.05 and exact p values were written when a trend was observed (p < 0.1). Exact two-way ANOVA p values and the number of animals are also displayed in Additional file 2: Tables S2 and S3, respectively.

Results

Body weight, food intake and running wheel activity

As previously reported [1, 50], both female and male ABA mice exhibited a severe body weight loss compared to CT mice (Fig. 1A, andB). Indeed, body weight decreased from day 6 to day 11 and from day 6 to day 14 in female and male ABA mice, respectively. In female mice only, the body weight further increased at days 15 and 16 compared to day 11. On day 16, males and females exhibited 17.2% and 13.2% of body weight loss, respectively, compared to day 5 (before the limited access to food). Body weight loss was associated with a reduced food intake in ABA mice compared to CT mice (Fig. 1C, andD).

Fig. 1
figure 1

Body weight and food intake. Body weight (A and B) and food intake (C and D) measured in female (open circles) and male (open squares) mice submitted to the activity-based anorexia (ABA) model or in control mice (CT). *, p < 0.05 vs. CT

Full size image

During the first 5 days (no limitation of food access), both male and female mice exhibited running wheel activity during the dark phase (Fig. 2B, C). After food access restriction, physical activity during the dark phase remained unchanged until day 7/8 and then decreased in both male and female mice (Fig. 2B). Of note, the level of physical activity during the dark phase remained higher in female than in male mice. By contrast, physical activity during the light phase increased from day 6 in both sexes, although this increase was higher in male mice compared to female mice (Fig. 2C) that is mainly due to the appearance of food-anticipatory activity (Fig. 2D). At the end of the ABA protocol, female mice showed higher wheel activity during the dark phase than male mice (Fig. 2D).

Fig. 2
figure 2

Running wheel activities. Running wheel activities (km) were measured in female (open circles) and male (open squares) mice submitted to the activity-based anorexia (ABA) model. A Running wheel activity measured at day 5. B Running wheel activity measured during the dark phase from day 1 to day 16. *p < 0.05 vs. day 5. C Running wheel activity measured during the light phase from day 1 to day 16. *, p < 0.05 vs. day 5. D Running wheel activity measured at day 16. #, p < 0.05 vs. males. In each graph, significant two-way ANOVA p values for the time (Hour), the sex (Sex) or for interaction (Int) are shown by bold and underlined text (n = 11–12/group)

Full size image

Central clock genes expression in female and male mice in response to ABA model

Expression of clock genes was studied in the hypothalamus area containing the SCN (Fig. 3). In females, Per1 (p < 0.05), Per2 (p < 0.01), Cry1 (p < 0.05) and Cry2 (p < 0.01) mRNA levels were significantly increased at EoR compared to EoA in ABA mice, but not in CT mice (Fig. 3). Rev-erbα mRNA expression was similarly modified in CT and ABA mice between EoA and EoR. By contrast, there was no significant changes in Bmal1 and Clock mRNA level according to the circadian phases, even if a trend for a reduction of Bmal1 mRNA level at EoA was observed in ABA mice compared to CT mice (p = 0.083). In male mice, Bmal1 mRNA expression was significantly different between EoR and EoA (two-way ANOVA p valueCR < 0.05; Fig. 3) but was not affected by the ABA procedure. Thus, our data underline that only female mice exhibited significant alterations of clock genes expression in the SCN in response to the ABA model.

Fig. 3
figure 3

Clock genes expression in the suprachiasmatic nucleus. Clock genes expression (Rev-erbα, Bmal1, Clock, Per1, Per2, Cry1 and Cry2) was quantified in the suprachiasmatic nucleus (SCN) in female (open circles) and male (open squares) mice submitted to the activity-based anorexia (ABA) model or in control mice (CT), at the end of active phase (EoA) or resting phase (EoR). In each graph, significant two-way ANOVA p values for the ABA model (ABA), the circadian rhythm (CR) or for interaction (Int) are shown by bold and underlined text (ABA, CR or Int). *, p < 0.05 and **, p < 0.01 with post-tests (n = 5–6 per group)

Full size image

Peripheral clock genes expression in female and male mice in response to ABA mode

Then, we evaluated expression of clock genes in peripheral tissues, such as ileum, colon and liver. Interestingly, we observed a sex-dependent response to the ABA model. Indeed, in the ileum, statistical analysis (two-way ANOVA p values and post-tests) revealed that all studied factors, namely, Bmal1, Clock, Per1, Per2, Cry1, Cry2 and Rev-erbα showed altered mRNA levels in female ABA mice (for all factors, two-way ANOVA p valueABA < 0.05). In males, only Per2 and Cry1 mRNA levels were affected by ABA: a circadian variation of Per2 mRNA expression was observed in male ABA mice (p < 0.0001) that was not observed in CT (Fig. 4). By contrast, the circadian variation observed for Cry1 in male CT mice (p < 0.001) disappeared in ABA mice (Fig. 4). In the colon, ABA induced alterations in Per2, Cry1 and Cry2 mRNA levels in females. Indeed, female ABA mice exhibited a variation of Per2 (p < 0.0001) and Cry2 (p < 0.001) expression between EoA and EoR, that was not observed in female CT mice (Fig. 5). By contrast, for Cry1, the differences observed in CT mice (p < 0.001) was abolished in ABA mice (Fig. 5). Similarly, male CT mice exhibited a significant modification for Cry1 between EoA and EoR (p < 0.05) that was not present in ABA mice. In addition, male ABA mice at EoR exhibited trends for slight modifications for Per2 (p = 0.052) and Rev-erbα (p = 0.072) expression levels compared to CT mice at EoR. Thus, ABA increased the amplitude of Cry2 mRNA variation but decreased the amplitude of Cry1 mRNA variation in both the ileum and colon of female mice. By contrast, in male mice, ABA only decreased the amplitude of Cry1 mRNA variation.

Fig. 4
figure 4

Clock genes expression in the ileum. Clock genes expression (Rev-erbα, Bmal1, Clock, Per1, Per2, Cry1 and Cry2) was quantified in the ileum in female (open circles) and male (open squares) mice submitted to the activity-based anorexia (ABA) model or in control mice (CT), at the end of active phase (EoA) or resting phase (EoR). In each graph, significant two-way ANOVA p values for the ABA model (ABA), the circadian rhythm (CR) or for interaction (Int) are shown by bold and underlined text (ABA, CR or Int). *, p < 0.05; **, p < 0.01; ***, p < 0.001 and ****, p < 0.0001 with post-tests (n = 7–12 per group)

Full size image
Fig. 5
figure 5

Clock genes expression in the colon. Clock genes expression (Rev-erbα, Bmal1, Clock, Per1, Per2, Cry1 and Cry2) was assessed in the colon in female (open circles) and male (open squares) mice submitted to the activity-based anorexia (ABA) model or in control mice (CT), at the end of active phase (EoA) or resting phase (EoR). In each graph, significant two-way ANOVA p values for the ABA model (ABA), the circadian rhythm (CR) or for interaction (Int) are shown by bold and underlined text (ABA, CR or Int). *, p < 0.05; **, p < 0.01; ***, p < 0.001 and ****, p < 0.0001 with post-tests (n = 8–12 per group)

Full size image

In the liver, while all studied clock genes showed no changes in response to ABA model in females, except a modest effect for Per1 (two-way ANOVA p valueABA < 0.05), ABA procedure affected Clock, Per2, Cry1, Cry2 and Rev-erbα mRNA expression in males (Fig. 6). Indeed, male ABA mice exhibited significant circadian mRNA levels modification for Per2 (p < 0.001) and Cry2 (p < 0.01) that was not present in CT mice. By contrast, the difference observed in CT mice for Cry1 (p < 0.001) disappeared in ABA mice. Finally, ABA mice exhibited significant differences for Clock (at EoA, p < 0.05) and Rev-erbα (at EoR, p < 0.001) compared to CT mice (Fig. 6).

Fig. 6
figure 6

Clock genes expression in the liver. Clock genes expression (Rev-erbα, Bmal1, Clock, Per1, Per2, Cry1 and Cry2) was quantified in the liver in female (open circles) and male (open squares) mice submitted to the activity-based anorexia (ABA) model or in control mice (CT), at the end of active phase (EoA) or resting phase (EoR). In each graph, significant two-way ANOVA p values for the ABA model (ABA), the circadian rhythm (CR) or for interaction (Int) are shown by bold and underlined text (ABA, CR or Int). *, p < 0.05; **, p < 0.01; ***, p < 0.001 and ****, p < 0.0001 with post-tests (n = 9–12 per group)

Full size image

Interestingly, in all peripheral studied tissues, ABA increased the amplitude of Per2 in both males and females

To resume, the ABA model per se induced sex- and tissue-dependent alterations of circadian clock genes expression, e.g., major effects on liver for males and on ileum for females.

Analysis of gut–microbiota according to circadian rhythm in female and male ABA mice

We then studied gut–microbiota composition by focusing on bacterial phyla and families that we had previously reported as changed during the ABA model [7]. We first focused on specific bacterial phyla and observed a sex-dependent response (Fig. 7). Indeed, in female mice, Eubacteria abundance remained unaffected in response to the circadian rhythm and the ABA model. By contrast, in males, Eubacteria abundance varied between EoA and EoR but only in response to ABA (p < 0.0001). The Firmicutes/Bacteroidetes ratio changed in response to the circadian rhythm in females (two-way ANOVA, p valueCR = 0.0004), while it was not affected in males. In males, Firmicutes and Bacteroidetes abundance changed during the circadian rhythm (two-way ANOVA p valueCR < 0.05) and the ABA model amplified these variations to reach significant differences between ABA–EoA and ABA–EoR (p < 0.001 and p < 0.05 for Firmicutes and Bacteroidetes, respectively). Again, for Gram-negative bacteria, we noticed a sex-dependent response (Fig. 8). We then focused on variations in bacterial family abundances. In female mice, we observed a circadian variation of Prevotella and γ-proteobacteria abundance in CT (p < 0.05 and p < 0.001, respectively) that did not reach significance in response to ABA model (Fig. 8). By contrast, in males, while β-proteobacteria and δ-proteobacteria did not show circadian variation in CT mice, ABA mice exhibited a significant circadian variation of these bacteria (p < 0.0001 and p < 0.001, respectively; Fig. 8). Concerning the Gram-positive bacteria, the ABA model per se induced an increase in Lactobacillus in both female and male mice, particularly L. reuteri, L. murinus/animalis and L. johnsoni/gasseri (two-way ANOVA, p valueABA < 0.05). We did not observe a circadian variation of Lactobacillus, except for L. johnsoni/gasseri in females (two-way ANOVA p valueCR = 0.0204; Fig. 9). Thus, the increase in Lactobacillus observed in response to ABA is both sex and circadian rhythm independent (Fig. 9). By contrast, in response to ABA, Clostridium cocleatum showed a circadian variation in males (p < 0.001, Fig. 9) that was not observed in CT mice.

Fig. 7
figure 7

Relative abundance of Eubacteria, Firmicutes and Bacteroidetes in the cecal content. Relative abundance of Eubateria, Firmicutes and Bacteroidetes was quantified in the cecal content of female (open circles) and male (open squares) mice submitted to activity-based anorexia (ABA) model or in control mice (CT), at the end of active phase (EoA) or resting phase (EoR). In each graph, significant two-way ANOVA p values for the ABA model (ABA), the circadian rhythm (CR) or for interaction (Int) are shown by bold and underlined text (ABA, CR or Int). *, p < 0.05; ***, p < 0.001 and ****, p < 0.0001 with post-tests (n = 8–12 per group)

Full size image
Fig. 8
figure 8

Relative abundance of Gram negative bacteria in the cecal content. Relative abundance of β-Proteobacteria, δ-Proteobacteria, γ-Proteobacteria, Prevotella, Verrucomicrobia and Akkermansia mucinipila was quantified in the cecal content of female (open circles) and male (open squares) mice submitted to the activity-based anorexia (ABA) model or in control mice (CT), at the end of active phase (EoA) or resting phase (EoR). In each graph, significant two-way ANOVA p values for the ABA model (ABA), the circadian rhythm (CR) or for interaction (Int) are shown by bold and underlined text (ABA, CR or Int). *, p < 0.05; ***, p < 0.001 and ****, p < 0.0001 with post-tests (n = 7–12 per group)

Full size image
Fig. 9
figure 9

Relative abundance of Gram positive bacteria in the cecal content. Relative abundance of Clostridium cocleatum, total Lactobacillus, L. reuteri, L. johnsonii/gasseri, L. murinus/animalis, Roseburia and Faecalibacterium prausnitzii was quantified in the cecal content of female (open circles) and male (open squares) mice submitted to the activity-based anorexia (ABA) model or in control mice (CT), at the end of active phase (EoA) or resting phase (EoR). In each graph, significant two-way ANOVA p values—for the ABA model (ABA), the circadian rhythm (CR) or for interaction (Int) are shown by bold and underlined text (ABA, CR or Int). *, p < 0.05; ***, p < 0.001 and ****, p < 0.0001 with post-tests (n = 7–12 per group)

Full size image

Discussion

In the present study, we report for the first time alterations in clock genes expression at both central and peripheral levels in response to the activity-based anorexia (ABA) model (Fig. 10), which is considered to be the most relevant animal model to study the pathophysiology of anorexia nervosa (AN) [43, 44]. In addition, we also show that ABA mice exhibit circadian variations of gut–microbiota composition (Fig. 11). Finally, we emphasize that all these factors are modulated in a sex-dependent manner.

Fig. 10
figure 10

Summary of the effects of the activity-based anorexia (ABA) model on female and male mice clock genes expression in both suprachiasmatic nucleus (SCN) and peripheral tissues. The effects of ABA are highlighted in blue according to the statistical analysis (two-way ANOVA with Tukey post-tests, see statistical methods). Symbols represent: no circadian variation ( ), circadian variation with low ( ), moderate ( ) or large amplitude ( ), reduced (↓) or increased expression (↑)

Full size image
Fig. 11
figure 11

Summary of the effects of the activity-based anorexia (ABA) model on female and male mice gut–microbiota composition. The effects of ABA are highlighted in blue according to the statistical analysis (two-way ANOVA with Tukey post-tests, see statistical methods). Symbols represent: no circadian variation ( ), circadian variation with low ( ), moderate ( ) or large amplitude ( ), reduced (↓) or increased expression (↑)

Full size image

Patients with AN frequently show sleep disorders [2, 3, 31] and circadian hormonal dysregulations [23, 26] that may contribute to the AN pathophysiology, which remains not completely understood. During the last decade, the role of the microbiota–gut–brain axis has emerged in the regulation of feeding behavior [15, 41]. In the ABA model, previous studies reported altered nycthemeral changes with the appearance of physical activity during the light phase [1, 29]. Interestingly this diurnal activity, that mainly corresponds to FAA, is more marked in males compared to females [1]. Li et al. have underlined the role of gonadal hormones, since FAA differences induced by restriction feeding were abolished in gonadectomized animals [33]. In the present study, we also observed increased diurnal physical activity that was more marked in male mice. However, until now, no study evaluated the regulation of clock genes expression in mice submitted to the ABA model.

As described in the clock gene database, CircaDB, we observed that clock genes have a specific tissue expression [39]. We focused on the SCN, the master clock mainly controlled by light/dark cycle, and on peripheral tissues: liver, ileum and colon. Interestingly, ABA induced an alteration of clock genes expression in the SCN only in females. Particularly, our data suggest an increase of Per1, Per2, Cry1 and Cry2 variations between the end of resting and active phases. In males, we did not observe these changes in response to ABA. Previous studies showed sex-dependent regulation of the clock genes expression in the SCN in response to stress or corticoids [11, 36], while both ABA and AN patients showed activation of the corticotrope axis [4]. Whether these alterations of clock genes expression in the SCN contribute to the higher prevalence of AN in women would deserve further investigations.

In peripheral tissues, ABA mice also exhibited modifications of clock genes expression in a sex-dependent manner. Indeed, in response to ABA model, clock genes expression was more markedly affected in female mice in the ileum, by contrast to the liver, where changes are more pronounced in males. Interestingly, Heddes et al. reported that a disruption of the intestinal circadian rhythm by Bmal1 intestinal knock-out induced changes in gut–microbiota composition and metabolome [25]. It is now well-established that a gut–microbiota dysbiosis occurs in both AN patients and ABA mice with, in both cases, an increase in Lactobacillus levels [5, 7, 20]. In the present study, we confirm the gut dysbiosis in ABA mice compared to CT but we also highlight circadian variations of gut–bacteria species that are amplified in response to ABA model, particularly in males. Even if the gut dysbiosis observed in male ABA mice and female rats may mainly be due to reduced food intake [7, 52], the putative role of intestinal clock genes disturbances on the gut–microbiota needs to be further explored.

Gut–microbiota may affect clock genes expression in both intestinal and extra-intestinal cells [54]. Indeed, germ-free mice exhibited altered intestinal clock genes expression [53] and short chain fatty acids, which are byproducts of bacterial fiber fermentation, were shown to regulate Bmal1 and Per2 in hepatocytes [32]. In this study, clock genes expression in the liver was more pronounced in ABA males that also exhibited greater alterations in gut–microbiota circadian variations compared to females.

Clock genes are finally known to regulate several pathways, such as TLR4 expression and inflammatory responses. For instance, a circadian regulation of TLR4 expression has been described in the colon and the SCN [39]. TLR4 circadian expression is regulated by Bmal1 in macrophages [38] or by RORα and REV–ERBα in intestinal epithelial cells [35]. Further experiments should decipher the role of intestinal clock genes dysregulation on intestinal TLR4 expression, since we previously reported that TLR4 pathway is affected [4] and contributes to the microbiota–gut–brain axis in response to the ABA model [50].

The present study presents several limitations. We used C57BL/6 mice that are deficient in melatonin [42], an important effector of the circadian clock. However, C57BL/6 mice are commonly used for studies on circadian rhythm [19, 28, 39] and have been reported as good responders to ABA [40]. In a recent study in C57BL/6 mice, melatonin injections improved ethanol-induced liver injury by reprogramming the circadian protein BMAL1 [56]. It could be of interest to further evaluate the impact of melatonin on ABA model in C57BL/6 mice. In the present study, to limit the number of animals, we have chosen to limit our evaluation at two timepoints and to compare the circadian clock gene expression in ABA mice to control mice (free access to food and no activity wheel). Further research should also include additional timepoints to obtain a complete kinetic profile. It could be also of interest to compare the data to other groups, such as mice with restricted access to food and no activity wheel or mice with free access to food and to an activity wheel, to evaluate the role of starvation and physical activity, respectively. The impact of food on circadian rhythm has been reviewed [8], as well as the impact of physical activity in humans [46].

Perspective and significance

To conclude, we report here for the first time a sex-dependent alteration of clock genes expression in the ABA model in both central and peripheral tissues, as well as a modulation of circadian fluctuations in gut–microbiota composition. Our data suggest that an altered circadian rhythm may contribute to the dysfunction of the microbiota–gut–brain axis observed in patients with AN. Further experiments should decipher the underlying mechanisms leading to altered central and intestinal clock genes expression, as well as gut dysbiosis, in anorectic conditions.

Availability of data and materials

The data sets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ABA:

Activity-based anorexia

AN:

Anorexia nervosa

Bmal1:

Basic helix–loop–helix ARNT-like 1

Clock:

Clock circadian regulator

CR:

Circadian rhythm

Cry1:

Cryptochrome circadian regulator 1

Cry2:

Cryptochrome circadian regulator 2

CT:

Control mice

EoA:

End of activity phase

EoR:

End of resting phase

FAA:

Food anticipatory activity

Gapdh:

Glyceraldehyde-3-phosphate dehydrogenase

Int:

Interaction effect of two-way ANOVA

Per1:

Period circadian regulator 1

Per2:

Period circadian regulator 2

Rev-erbα (newly named Nr1d1):

Nuclear receptor subfamily 1 group D member 1

SCN:

Suprachiasmatic nucleus

References

  1. Achamrah N, Nobis S, Goichon A, Breton J, Legrand R, do Rego JL, do Rego JC, Déchelotte P, Fetissov SO, Belmonte L, Coëffier M. Sex differences in response to activity-based anorexia model in C57Bl/6 mice. Physiol Behav. 2017;170:1–5. https://doi.org/10.1016/j.physbeh.2016.12.014.

    Article  CAS  PubMed  Google Scholar 

  2. Allison KC, Spaeth A, Hopkins CM. Sleep and eating disorders. Curr Psychiatry Rep. 2016;18:92. https://doi.org/10.1007/s11920-016-0728-8.

    Article  PubMed  Google Scholar 

  3. Asaad Abdou T, Esawy HI, Abdel Razek Mohamed G, Hussein Ahmed H, Elhabiby MM, Khalil SA, El-Hawary YA. Sleep profile in anorexia and bulimia nervosa female patients. Sleep Med. 2018;48:113–6. https://doi.org/10.1016/j.sleep.2018.03.032.

    Article  PubMed  Google Scholar 

  4. Belmonte L, Achamrah N, Nobis S, Guérin C, Riou G, Bôle-Feysot C, Boyer O, Richard V, Rego JCD, Déchelotte P, Goichon A, Coëffier M. A role for intestinal TLR4-driven inflammatory response during activity-based anorexia. Sci Rep. 2016;6:35813. https://doi.org/10.1038/srep35813.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Breton J, Déchelotte P, Ribet D. Intestinal microbiota and anorexia nervosa. Clin Nutr Exp. 2019. https://doi.org/10.1016/j.yclnex.2019.05.001.

    Article  Google Scholar 

  6. Breton J, Legrand R, Achamrah N, Chan P, do Rego JL, do Rego JC, Coëffier M, Déchelotte P, Fetissov SO. Proteome modifications of gut microbiota in mice with activity-based anorexia and starvation: role in ATP production. Nutrition. 2019;67–68: 110557. https://doi.org/10.1016/j.nut.2019.110557.

    Article  CAS  PubMed  Google Scholar 

  7. Breton J, Tirelle P, Hasanat S, Pernot A, L’Huillier C, de Rego J-C, Déchelotte P, Coëffier M, Bindels LB, Ribet D. Gut microbiota alteration in a mouse model of anorexia nervosa. Clin Nutr. 2020. https://doi.org/10.1016/j.clnu.2020.05.002.

    Article  PubMed  Google Scholar 

  8. Cai Y, Liu Y, Wu Z, Wang J, Zhang X. Effects of diet and exercise on circadian rhythm: role of gut microbiota in immune and metabolic systems. Nutrients. 2023;15:2743. https://doi.org/10.3390/nu15122743.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Call C, Walsh BT, Attia E. From DSM-IV to DSM-5: changes to eating disorder diagnoses. Curr Opin Psychiatry. 2013;26:532–6. https://doi.org/10.1097/YCO.0b013e328365a321.

    Article  PubMed  Google Scholar 

  10. Chomczynski P, Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on. Nat Protoc. 2006;1:581–5. https://doi.org/10.1038/nprot.2006.83.

    Article  CAS  PubMed  Google Scholar 

  11. Chun LE, Christensen J, Woodruff ER, Morton SJ, Hinds LR, Spencer RL. Adrenal-dependent and -independent stress-induced Per1 mRNA in hypothalamic paraventricular nucleus and prefrontal cortex of male and female rats. Stress. 2018;21:69–83. https://doi.org/10.1080/10253890.2017.1404571.

    Article  PubMed  Google Scholar 

  12. Coëffier M, Miralles-Barrachina O, Le Pessot F, Lalaude O, Daveau M, Lavoinne A, Lerebours E, Déchelotte P. Influence of glutamine on cytokine production by human gut in vitro. Cytokine. 2001;13:148–54. https://doi.org/10.1006/cyto.2000.0813.

    Article  CAS  PubMed  Google Scholar 

  13. Daimon CM, Hentges ST. β-endorphin differentially contributes to food anticipatory activity in male and female mice undergoing activity-based anorexia. Physiol Rep. 2021;9: e14788. https://doi.org/10.14814/phy2.14788.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Davis C, Katzman DK, Kaptein S, Kirsh C, Brewer H, Kalmbach K, Olmsted MF, Woodside DB, Kaplan AS. The prevalence of high-level exercise in the eating disorders: etiological implications. Compr Psychiatry. 1997;38:321–6. https://doi.org/10.1016/S0010-440X(97)90927-5.

    Article  CAS  PubMed  Google Scholar 

  15. de Wouters d’Oplinter A, Huwart SJP, Cani PD, Everard A. Gut microbes and food reward: from the gut to the brain. Front Neurosci. 2022;16: 947240. https://doi.org/10.3389/fnins.2022.947240.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Deaver JA, Eum SY, Toborek M. Circadian disruption changes gut microbiome taxa and functional gene composition. Front Microbiol. 2018;9:737. https://doi.org/10.3389/fmicb.2018.00737.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Dominique M, Legrand R, Galmiche M, Azhar S, Deroissart C, Guérin C, do Rego J-L, Leon F, Nobis S, Lambert G, Lucas N, Déchelotte P. Changes in microbiota and bacterial protein caseinolytic peptidase B during food restriction in mice: relevance for the onset and perpetuation of anorexia nervosa. Nutrients. 2019;11:2514. https://doi.org/10.3390/nu11102514.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Duclos M, Ouerdani A, Mormède P, Konsman JP. Food restriction-induced hyperactivity: addiction or adaptation to famine? Psychoneuroendocrinology. 2013;38:884–97. https://doi.org/10.1016/j.psyneuen.2012.09.012.

    Article  PubMed  Google Scholar 

  19. Eckel-Mahan K, Sassone-Corsi P. Phenotyping circadian rhythms in mice. Curr Protoc Mouse Biol. 2015;5:271–81. https://doi.org/10.1002/9780470942390.mo140229.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Fan Y, Støving RK, Berreira Ibraim S, Hyötyläinen T, Thirion F, Arora T, Lyu L, Stankevic E, Hansen TH, Déchelotte P, Sinioja T, Ragnarsdottir O, Pons N, Galleron N, Quinquis B, Levenez F, Roume H, Falony G, Vieira-Silva S, Raes J, Clausen L, Telléus GK, Bäckhed F, Oresic M, Ehrlich SD, Pedersen O. The gut microbiota contributes to the pathogenesis of anorexia nervosa in humans and mice. Nat Microbiol. 2023. https://doi.org/10.1038/s41564-023-01355-5.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Gabloffsky T, Gill S, Staffeld A, Salomon R, Power Guerra N, Joost S, Hawlitschka A, Kipp M, Frintrop L. Food restriction in mice induces food-anticipatory activity and circadian-rhythm-related activity changes. Nutrients. 2022;14:5252. https://doi.org/10.3390/nu14245252.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Galmiche M, Déchelotte P, Lambert G, Tavolacci MP. Prevalence of eating disorders over the 2000–2018 period: a systematic literature review. Am J Clin Nutr. 2019;109:1402–13. https://doi.org/10.1093/ajcn/nqy342.

    Article  PubMed  Google Scholar 

  23. Galusca B, Prévost G, Germain N, Dubuc I, Ling Y, Anouar Y, Estour B, Chartrel N. Neuropeptide Y and α-MSH circadian levels in two populations with low body weight: anorexia nervosa and constitutional thinness. PLoS ONE. 2015;10: e0122040. https://doi.org/10.1371/journal.pone.0122040.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Goodwin-Groen S, Dong Y, Aoki C. Three daily intraperitoneal injections of sub-anesthetic ketamine ameliorate activity-based anorexia vulnerability of adult female mice. Int J Eat Disord. 2023. https://doi.org/10.1002/eat.24036.

    Article  PubMed  Google Scholar 

  25. Heddes M, Altaha B, Niu Y, Reitmeier S, Kleigrewe K, Haller D, Kiessling S. The intestinal clock drives the microbiome to maintain gastrointestinal homeostasis. Nat Commun. 2022;13:6068. https://doi.org/10.1038/s41467-022-33609-x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Herpertz S, Wagner R, Albers N, Blum WF, Pelz B, Langkafel M, Köpp W, Henning A, Oberste-Berghaus C, Mann K, Senf W, Hebebrand J. Circadian plasma leptin levels in patients with anorexia nervosa: relation to insulin and cortisol. Horm Res Paediatr. 1998;50:197–204. https://doi.org/10.1159/000023274.

    Article  CAS  Google Scholar 

  27. Hoek H. Incidence, prevalence and mortality of anorexia nervosa and other eating disorders. Curr Opin Psychiatry. 2006;19:389–94. https://doi.org/10.1097/01.yco.0000228759.95237.78.

    Article  PubMed  Google Scholar 

  28. Hughes ME, DiTacchio L, Hayes KR, Vollmers C, Pulivarthy S, Baggs JE, Panda S, Hogenesch JB. Harmonics of circadian gene transcription in mammals. PLoS Genet. 2009;5: e1000442. https://doi.org/10.1371/journal.pgen.1000442.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Jésus P, Ouelaa W, François M, Riachy L, Guérin C, Aziz M, Do Rego J-C, Déchelotte P, Fetissov SO, Coëffier M. Alteration of intestinal barrier function during activity-based anorexia in mice. Clin Nutr. 2014;33:1046–53. https://doi.org/10.1016/j.clnu.2013.11.006.

    Article  CAS  PubMed  Google Scholar 

  30. Kadota A, Iwakoshi-Ukena E, Fukumura K, Shikano K, Narimatsu Y, Furumitsu M, Ukena K. Effects of irregular feeding on the daily fluctuations in mRNA expression of the neurosecretory protein GL and neurosecretory protein GM genes in the mouse hypothalamus. IJMS. 2021;22:2109. https://doi.org/10.3390/ijms22042109.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Langlet B, Vestermark F, Stolt J, Zandian M, Södersten P, Bergh C. Physical activity and sleep during the first week of anorexia nervosa inpatient care. PLoS ONE. 2021;16: e0260077. https://doi.org/10.1371/journal.pone.0260077.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Leone V, Gibbons SM, Martinez K, Hutchison AL, Huang EY, Cham CM, Pierre JF, Heneghan AF, Nadimpalli A, Hubert N, Zale E, Wang Y, Huang Y, Theriault B, Dinner AR, Musch MW, Kudsk KA, Prendergast BJ, Gilbert JA, Chang EB. Effects of diurnal variation of gut microbes and high-fat feeding on host circadian clock function and metabolism. Cell Host Microbe. 2015;17:681–9. https://doi.org/10.1016/j.chom.2015.03.006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Li Z, Wang Y, Sun KK, Wang K, Sun ZS, Zhao M, Wang J. Sex-related difference in food-anticipatory activity of mice. Horm Behav. 2015;70:38–46. https://doi.org/10.1016/j.yhbeh.2015.02.004.

    Article  CAS  PubMed  Google Scholar 

  34. Montagner A, Korecka A, Polizzi A, Lippi Y, Blum Y, Canlet C, Tremblay-Franco M, Gautier-Stein A, Burcelin R, Yen Y-C, Je HS, Maha A-A, Mithieux G, Arulampalam V, Lagarrigue S, Guillou H, Pettersson S, Wahli W. Hepatic circadian clock oscillators and nuclear receptors integrate microbiome-derived signals. Sci Rep. 2016;6:20127. https://doi.org/10.1038/srep20127.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Mukherji A, Kobiita A, Ye T, Chambon P. Homeostasis in intestinal epithelium is orchestrated by the circadian clock and microbiota cues transduced by TLRs. Cell. 2013;153:812–27. https://doi.org/10.1016/j.cell.2013.04.020.

    Article  CAS  PubMed  Google Scholar 

  36. Murray A, Tharmalingam S, Khurana S, Lalonde C, Nguyen P, Tai TC. Effect of prenatal glucocorticoid exposure on circadian rhythm gene expression in the brains of adult rat offspring. Cells. 2022;11:1613. https://doi.org/10.3390/cells11101613.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Nobis S, Achamrah N, Goichon A, L’Huillier C, Morin A, Guérin C, Chan P, do Rego JL, do Rego JC, Vaudry D, Déchelotte P, Belmonte L, Coëffier M. Colonic mucosal proteome signature reveals reduced energy metabolism and protein synthesis but activated autophagy during anorexia-induced malnutrition in mice. Proteomics. 2018;18:1700395. https://doi.org/10.1002/pmic.201700395.

    Article  CAS  Google Scholar 

  38. Oishi Y, Hayashi S, Isagawa T, Oshima M, Iwama A, Shimba S, Okamura H, Manabe I. Bmal1 regulates inflammatory responses in macrophages by modulating enhancer RNA transcription. Sci Rep. 2017;7:7086. https://doi.org/10.1038/s41598-017-07100-3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Pizarro A, Hayer K, Lahens NF, Hogenesch JB. CircaDB: a database of mammalian circadian gene expression profiles. Nucleic Acids Res. 2013;41:D1009–13. https://doi.org/10.1093/nar/gks1161.

    Article  CAS  PubMed  Google Scholar 

  40. Pjetri E, de Haas R, de Jong S, Gelegen C, Oppelaar H, Verhagen LAW, Eijkemans MJC, Adan RA, Olivier B, Kas MJ. Identifying predictors of activity based anorexia susceptibility in diverse genetic rodent populations. PLoS ONE. 2012;7: e50453. https://doi.org/10.1371/journal.pone.0050453.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Romaní-Pérez M, Bullich-Vilarrubias C, López-Almela I, Liébana-García R, Olivares M, Sanz Y. The microbiota and the gut-brain axis in controlling food intake and energy homeostasis. Int J Mol Sci. 2021;22:5830. https://doi.org/10.3390/ijms22115830.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Roseboom PH, Namboodiri MAA, Zimonjic DB, Popescu NC, Rodriguez RI, Gastel JA, Klein DC. Natural melatonin `knockdown’ in C57BL/6J mice: rare mechanism truncates serotonin N-acetyltransferase1 Published on the World Wide Web on 9 November 1998.1. Mol Brain Res. 1998;63:189–97. https://doi.org/10.1016/S0169-328X(98)00273-3.

    Article  CAS  PubMed  Google Scholar 

  43. Schalla MA, Stengel A. Activity based anorexia as an animal model for anorexia nervosa–a systematic review. Front Nutr. 2019. https://doi.org/10.3389/fnut.2019.00069.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Scharner S, Stengel A. Animal models for anorexia nervosa—a systematic review. Front Hum Neurosci. 2021. https://doi.org/10.3389/fnhum.2020.596381.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Segers A, Depoortere I. Circadian clocks in the digestive system. Nat Rev Gastroenterol Hepatol. 2021. https://doi.org/10.1038/s41575-020-00401-5.

    Article  PubMed  Google Scholar 

  46. Shen B, Ma C, Wu G, Liu H, Chen L, Yang G. Effects of exercise on circadian rhythms in humans. Front Pharmacol. 2023;14:1282357. https://doi.org/10.3389/fphar.2023.1282357.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Teichman EM, O’Riordan KJ, Gahan CGM, Dinan TG, Cryan JF. When rhythms meet the blues: circadian interactions with the microbiota-gut-brain axis. Cell Metab. 2020;31:448–71. https://doi.org/10.1016/j.cmet.2020.02.008.

    Article  CAS  PubMed  Google Scholar 

  48. Timko CA, DeFilipp L, Dakanalis A. Sex differences in adolescent anorexia and bulimia nervosa: beyond the signs and symptoms. Curr Psychiatry Rep. 2019;21:1. https://doi.org/10.1007/s11920-019-0988-1.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Tirelle P, Breton J, Kauffmann A, Bahlouli W, L’Huillier C, Salameh E, Amamou A, Jarbeau M, Guérin C, Goichon A, do Rego J-C, Déchelotte P, Ribet D, Coëffier M. Gut microbiota depletion affects nutritional and behavioral responses to activity-based anorexia model in a sex-dependent manner. Clin Nutr. 2021;40:2734–44. https://doi.org/10.1016/j.clnu.2021.04.014.

    Article  CAS  PubMed  Google Scholar 

  50. Tirelle P, Salaün C, Kauffmann A, Bôle-Feysot C, Guérin C, Huré M, Goichon A, Amamou A, Breton J, do Rego J-L, Déchelotte P, Achamrah N, Coëffier M. Intestinal epithelial toll-like receptor 4 deficiency modifies the response to the activity-based anorexia model in a sex-dependent manner: a preliminary study. Nutrients. 2022;14:3607. https://doi.org/10.3390/nu14173607.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Trinh S, Kogel V, Kneisel L, Müller-Limberger E, Herpertz-Dahlmann B, Beyer C, Seitz J. Gut microbiota and brain alterations after refeeding in a translational anorexia nervosa rat model. Int J Mol Sci. 2023;24:9496. https://doi.org/10.3390/ijms24119496.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Trinh S, Kogel V, Voelz C, Schlösser A, Schwenzer C, Kabbert J, Heussen N, Clavel T, Herpertz-Dahlmann B, Beyer C, Seitz J. Gut microbiota and brain alterations in a translational anorexia nervosa rat model. J Psychiatr Res. 2021;133:156–65. https://doi.org/10.1016/j.jpsychires.2020.12.030.

    Article  PubMed  Google Scholar 

  53. Weger BD, Gobet C, Yeung J, Martin E, Jimenez S, Betrisey B, Foata F, Berger B, Balvay A, Foussier A, Charpagne A, Boizet-Bonhoure B, Chou CJ, Naef F, Gachon F. The mouse microbiome is required for sex-specific diurnal rhythms of gene expression and metabolism. Cell Metab. 2019;29:362-382.e8. https://doi.org/10.1016/j.cmet.2018.09.023.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Xia Y, Ding X, Wang S, Ren W. Circadian orchestration of host and gut microbiota in infection. Biol Rev. 2022. https://doi.org/10.1111/brv.12898.

    Article  PubMed  Google Scholar 

  55. Zhang R, Lahens NF, Ballance HI, Hughes ME, Hogenesch JB. A circadian gene expression atlas in mammals: implications for biology and medicine. Proc Natl Acad Sci USA. 2014;111:16219–24. https://doi.org/10.1073/pnas.1408886111.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Zhao Y, Zhang R, Wang Z, Chen Z, Wang G, Guan S, Lu J. Melatonin prevents against ethanol-induced liver injury by mitigating ferroptosis via targeting brain and muscle ARNT-like 1 in mice liver and HepG2 cells. J Agric Food Chem. 2022;70:12953–67. https://doi.org/10.1021/acs.jafc.2c04337.

    Article  CAS  PubMed  Google Scholar 

  57. Zipfel S, Giel KE, Bulik CM, Hay P, Schmidt U. Anorexia nervosa: aetiology, assessment, and treatment. Lancet Psychiatry. 2015;2:1099–111. https://doi.org/10.1016/S2215-0366(15)00356-9.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

The authors gratefully thank Fatima LEON (SCAC platform) for her helpful technical assistance.

Funding

The study was co-supported by the Charles Nicolle foundation, by the Microbiome foundation, the Roquette foundation for health, by European Union and Normandie Regional Council. Europe gets involved in Normandie with European Regional Development Fund (ERDF). CS and LR received the support of the University of Rouen Normandie during his/her PhD. These funders did not participate in the design, implementation, analysis, and interpretation of the data.

Author information

Authors and Affiliations

  1. Univ Rouen Normandie, INSERM, Normandie Univ, ADEN UMR 1073, Nutrition Inflammation and Microbiota Gut Brain Axis, UFR Santé, 22 Boulevard Gambetta, 76183, Rouen Cedex, France

    Colin Salaün, Marine Courvalet, Léna Rousseau, Kévin Cailleux, Jonathan Breton, Christine Bôle-Feysot, Charlène Guérin, Marion Huré, Alexis Goichon, Pierre Déchelotte, David Ribet, Najate Achamrah & Moïse Coëffier

  2. Univ Rouen Normandie, Institute for Research and Innovation in Biomedicine (IRIB), 76000, Rouen, France

    Colin Salaün, Marine Courvalet, Léna Rousseau, Kévin Cailleux, Jonathan Breton, Christine Bôle-Feysot, Charlène Guérin, Marion Huré, Alexis Goichon, Jean-Claude do Rego, Pierre Déchelotte, David Ribet, Najate Achamrah & Moïse Coëffier

  3. Univ Rouen Normandie, Inserm, CNRS, Normandie Univ, HERACLES US 51 UAR 2026, Behavioural Analysis Platform SCAC, 76000, Rouen, France

    Jean-Claude do Rego

  4. Department of Nutrition, CHU Rouen, 76000, Rouen, France

    Pierre Déchelotte, Najate Achamrah & Moïse Coëffier

Authors
  1. Colin Salaün
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marine Courvalet
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Léna Rousseau
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kévin Cailleux
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jonathan Breton
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Christine Bôle-Feysot
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Charlène Guérin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Marion Huré
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Alexis Goichon
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jean-Claude do Rego
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Pierre Déchelotte
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. David Ribet
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Najate Achamrah
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Moïse Coëffier
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization, CS, NA, and MoC; CS, MaC, DR, NA and MoC, formal analysis; CS, MaC, LR, KC, JB, CBF, CG, MH and AG, investigation; CS and MoC, original draft preparation; LR, JCdR, DR, PD and NA writing review and editing.

Corresponding author

Correspondence to Moïse Coëffier.

Ethics declarations

Ethics approval and consent to participate

All experimental procedures were approved by an ethical committee (APAFIS#9473-2016072016008305 v6).

Consent for publication

All authors have read and agreed to the published version of the manuscript.

Competing interests

The authors declare no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Figure S1.

Experimental design.

Additional file 2: Table S1.

Analysable n (n) and retained n after outliers statistical test (ROUT 1%). Table S2. Exact two-way ANOVA p values (ABA X Euthanized time) of clocks genes expression and gut–microbiota abundances. Table S3. Exact Tukey's multiple comparisons test p values of clocks genes expression and gut–microbiota abundances.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Salaün, C., Courvalet, M., Rousseau, L. et al. Sex-dependent circadian alterations of both central and peripheral clock genes expression and gut–microbiota composition during activity-based anorexia in mice. Biol Sex Differ 15, 6 (2024). https://doi.org/10.1186/s13293-023-00576-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13293-023-00576-x

Biology of Sex Differences

ISSN: 2042-6410

Contact us