Novel ghrelin receptor modulator agrelax reduces chocolate intake in overeating rats subject to foot-shock stress

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BACKGROUND

Obesity is one of the leading causes of disability and death worldwide, affecting not only adults but also children and adolescents. The obesity prevalence is growing steadily and amounts to about 11% in men and 15% in women¹. Numerous studies have shown the bidirectional relationship between obesity and stress through cognitive, behavioral, physiological, and biochemical pathways [1–4]. Conversely, obesity itself becomes a stressor due to societal weight stigma [4, 5]. Stress impairs such cognitive functions as self-regulation and executive control, leading to unhealthy eating behavior, including binge eating (BED) and food addiction (FA) [6–8].

BED, classified in the fifth edition of Diagnostic and Statistical Manual of mental disorders, involves episodes of excessive eating with loss of control, often driven by emotional distress, body image concerns, and guilt, without compensatory behaviors [9–11]. FA, not officially registered yet, shares addictive-like symptoms such as cravings, withdrawal, and tolerance, measured by the Yale Food Addiction Scale [12–14]. Their clinical performances have similar symptoms, like compulsive overeating, but treatment approaches differ, with cognitive behavioral therapy often recommended for BED and substance-specific interventions for FA [7, 8].

Physiologically, stress activates the hypothalamic-pituitary-adrenal axis, increasing cortisol levels, which promotes abdominal fat deposition and appetite stimulation [3, 4]. Biochemically, stress alters hormones, influencing hunger and metabolism, such as leptin, neuropeptide Y, and ghrelin [1]. Ghrelin is the endogenous ligand for growth hormone secretagogue receptor 1a type (or ghrelin receptor), and takes part in different body processes, particularly in food intake regulation [15, 16]. Moreover, ghrelin was shown to stimulate reward seeking behavior specifically in conjunction with stress exposure [17, 18]. Thus, ghrelin receptor antagonists appear to be promising in treatment of obesity and addictive disorders.

Due to high selectivity (and therefore low toxicity), low immunogenicity, and cost-effective production compared to biologics, the popularity of peptide drugs in treatment of metabolic, oncologic, cardiovascular, and neurodegenerative diseases is growing [19–21]. The structural features of peptides determine their poor oral bioavailability and rapid degradation that can be overcome through structural modifications and selection of a suitable way of administration. For instance drug delivery for targeting brain disorders can be achieved by intranasal route, same as in the present investigation [22–24].

Aim

Based on the above, it is necessary to explore new strategies for obesity therapy amidst stress conditions. Therefore the aim of this research was to estimate the impact of novel peptide antagonist of the ghrelin receptor agrelax on rats eating behavior in the limited access to high palatable foods model under electric foot shock stress conditions.

Methods

Animals

The experiment involved 36 male Wistar rats raised in the Rappolovo nursery, Leningrad Region. The rats were kept in vivarium conditions in standard plastic cages with free access to water and food under inverted light from 8:00 to 20:00 at a temperature of 22 ± 2°C. The work with the animals was carried out in accordance with the “Rules of Laboratory Practice in the Russian Federation” (Order of the Ministry of Health of the Russian Federation of 2003, No. 267). After receipt from the nursery, the animals underwent a 2-week quarantine period in the corresponding block of the vivarium. The rats were then divided equally (n = 18) to participate in two experiments.

Model of limited access to high-calorie foods

The feeding behavior of rats was studied in a model of limited access to high-calorie food, which was a chocolate treat consisting of Nutella chocolate spread (Ferrero, Alba, Turin, Italy), crushed rat pellets (4RF18; Mucedola; Settimo Milanese) and water in the following weight ratio: 52% Nutella, 33% food pellets and 15% water. The caloric content of the diet was 3.63 kcal/g. Limited access was ensured by providing the chocolate treat on a “Monday” – “Wednesday” – “Friday” schedule for one hour, and besides, before being given out, for 15 minutes the rats only saw the treat and smelled the chocolate, but could not eat it. This model requires preliminary training to achieve a stable level of chocolate treat consumption — deviations of no more than 10% over 3 days. During the first week, rats are kept in cages in groups of 6 and eat the treat together. Then the animals are placed in cages individually, where chocolate treat eating without additional impacts continues for another 3 weeks [9, 11, 25].

Elevated plus maze test

Anxiety behavior of rats was assessed in the elevated plus maze test. The behavior of rats was studied in an elevated plus maze setup, which consisted of two open arms measuring 50 × 10 cm and two closed arms measuring 50 × 10 cm with an open top, located perpendicular to each other. The height above the floor was 1 m. The animal was placed in the center of the maze. By pressing the appropriate key on the “ethograph” connected to the computer, the time spent in the closed and open arms, the number of head-dippings from the platform, the number of transitions between and emerges from the closed arms were recorded. The test lasted 3 minutes [26].

Marble burying test

Marble burying test is proposed as a model of obsessive-compulsive disorder associated with compulsive ideas and actions. A 20 × 25 × 17 cm cage was covered with sawdust in a 5 cm layer, and 20 glass balls with a diameter of 1 cm were placed on top at equal distances. The rat was placed in the cage for 30 min. After this time, the number of balls covered by sawdust by more than 2/3 was counted. In this experiment, each animal was tested 3 times [27–29].

Electrical stimulation of the rat limbs

Electrical stimulation of the rat limbs, or Foot-shock (FS), was used to perform mild stress in rats. To carry out the stress exposure, the animal was placed in a special chamber designed by Institute of Experimental Medicine (Saint-Petersburg, Russia) engineers with an electrified floor, in which electrical stimulation of the limbs was performed with a current of 0.6 mA for 60 seconds [10].

Drugs

As a control for the drug effect, animals received a physiological saline (0.9% NaCl solution) intranasally, 10 µl in each nostril. Agrelax is a peptide analogue of a ghrelin antagonist consisting of 13 amino acid residues, including non-standard ones, which was synthesized in the Department of Neuropharmacology named after S.V. Anichkov, Institute of Experimental Medicine (Saint Petersburg, Russia). Agrelax was administered intranasally at a dosage of 1 mg/mL, 10 μL in each nostril [30].

Research design

The work consists of two experiments. The first experiment (validization) involved 18 rats divided into 2 groups: control with neither additional influences nor chocolate treat (Cv, n = 6) and experimental group (Ev, n = 12): rats trained using the model of limited access to high-calorie food. After the training of Ev (described earlier), the behavior of animals in both groups was studied using marble burying and elevated plus maze tests.

This second experiment (limited access to high-calorie foods under stress conditions) also included 18 rats, which were divided into groups as follows: control rats (C, n = 6), which did not received chocolate treat, and experimental rats (E, n = 12), which were trained in the model of limited access to high-calorie food, both were exposed to stress and drugs. FS was performed once a week one hour before feeding the chocolate treat. Agrelax and saline were administered 30 minutes after FS exposure, i. e. 30 minutes before feeding the treat. The effects of stress and agrelax on the consumption of a chocolate treat and the total amount eaten per day after exposure to stress (chow and chocolate) were assessed.

Statistical procedures

Statistical data processing and graphical visualization of the results were performed on the Python 3 programming language (https://www.anaconda.com/) employing the Jupyter Notebook platform (https://jupyter.org/). Fisher’s exact test was used to analyze contingency tables. The ANOVA test for related samples and paired t-Test with the correction for multiple comparisons were used to assess the statistical significance of the experimental results. All data were presented as the mean ± standard error of the mean (SEM). The difference in means was considered reliable at p < 0.05.

Results

It has been noted that rats respond differently to stress and administration of ghrelin receptor antagonist depending on the average amount of consumed chocolate treat. Thus, after exposure to stress, the amount of chocolate treat eaten increased only for rats that consumed more than 36 kcal/hour before the exposure. In the present study, anxiety and compulsivity were compared for three groups: intact control, which never consumed chocolate treat (Cv), rats with an average amount of chocolate treat consumed more (Ev_M, n = 6) and less (Ev_L, n = 6) 36 kcal.

The effect of chocolate treat consumption on the anxious behavior of rats was assessed in the elevated plus maze. Animals from the Ev_M group showed greater anxiety compared to the Ev_L group in terms of the percentage of time spent in the open arms (36.7 ± 1.5% and 45.8 ± 2.6%, respectively), while the indicators of the Cv group did not differ significantly from the experimental groups (35.2 ± 6.2%) (Fig. 1, a). The increased anxiety of rats from the Ev_M group (11.0 ± 0.4) relative to both the Cv (6.2 ± 0.9) and EvL (3.0 ± 0.9) groups is also evidenced by the quantity of emerges from the closed arms of the maze (Fig. 1, b). The assessment of the sum of head-dippings demonstrated lower exploratory activity of the Ev_M group (3.5 ± 0.2) relative to Ev_L (5.5 ± 0.2), but not Cv (2.2 ± 0.7) (Fig. 1, c). In addition, the total number of transitions between closed arms indicates a significantly higher locomotor activity of the Ev_M group: 1.9 ± 0.6 for Cv, 2.0 ± 0.9 for Ev_L and for 5.0 ± 0.4 Ev_M (Fig. 1, d).

 

Fig. 1. The elevated plus maze test results: a, the percentage of time spent in the open arms; b, the sum of emerges from the closed arms; c, the number of head-dippings from the platform; and the quantity of transitions between closed arms (d). Cv—control rats not receiving chocolate treat, Ev_L—rats, consumed less than 36 kcal treat per hour, Ev_M—rats, consumed more than 36 kcal treat per hour. Values are mean ± standard error of the mean, *p < 0.05.

 

The compulsive behavior of rats was assessed using the marble burying test. Rats from the Ev_M group had greater compulsivity than those from the Ev_L group, as evidenced by the number of buried marbles 11.0 ± 0.4 and 8.0 ± 0.9, respectively. Moreover, none of the experimental groups differed significantly from the control (9.8 ± 0.8) (Fig. 2). Suchwise, it was demonstrated that rats consuming more or less than 36 kcal of chocolate treat differ not only in response to FS, but also in their levels of anxiety and compulsivity.

 

Fig. 2. The number of buried balls in the marble burying test. Cv, control rats not receiving chocolate treat, Ev_L, rats, consumed less than 36 kcal treat per hour, Ev_M, rats, consumed more than 36 kcal treat per hour. Values are mean ± standard error of the mean; *p < 0.05.

 

In the present study, a relationship between the amount of chocolate consumed and the response to FS was also found (see tab. 1), and therefore the experimental group was divided into rats with an average amount of chocolate consumed greater than (E_M) and less than (E_L) 36 kcal. Stress exposure, as well as the intranasal injection of agrelax, did not have a significant effect on the total daily caloric intake for either group. FS significantly increased chocolate treat consumption by the E_M group from 47.9 ± 4.0 to 56.0 ± 5.4 kcal, the introduction of saline did not change the behavior of the animals (56.8 ± 6.1 kcal), and the administration of agrelax decreased the amount of chocolate treat consumed to the values obtained before the onset of stress exposure — 41.0 ± 3.6 kcal. Agrelax administration in the E_L group showed an increase in chocolate treat intake (31.7 ± 6.3 kcal) compared to the values obtained after FS, however, no significant differences in the average amount of chocolate treat eaten before (28.2 ± 3.8 kcal) and after (23.7 ± 4.9 kcal) stress were found (Fig. 3).

 

Table 1. Distribution of rats sensitive and insensitive to foot shock as a function of the average amount of the chocolate treat eaten

Mean treat eaten (kcal) / sensitivity to foot shock	Sensitive to foot shock	Insensitive to foot shock	Total
>36	5	0	5
<36	1	6	6
Total	6	6	12
Note. Chi-square test with Yates correction <0.05.

 

Fig. 3. The amount of chocolate treat consumed by rats in the limited access to high-caloric foods model. E_L, rats, consumed less than 36 kcal treat per hour, E_M, rats, consumed more than 36 kcal treat per hour. Values are mean ± standard error of the mean. *p < 0.05.

 

Discussion

Obesity and stress are becoming increasingly common today, while they contribute to the formation and maintenance of each other [4]. In this study, the authors examined the compulsive pattern of eating disorders, characteristic of such conditions as binge eating and food addiction [7, 8]. Both of these conditions are risk factors for the development of obesity, and their symptoms, in particular the compulsive desire to consume highly attractive food, are exacerbated by stress [6, 13].

Studies of both chemical and behavioral addictions indicate that only a part of the sample will be tropic to a certain chemical substance or action. Thus only 10.2% of excessive drinkers meet criteria for alcohol dependence, meaning that 90% engage in risky drinking without addiction [31]. Similarly, just 0.4% of cocaine users and 0.2% of heroin users develop dependence annually². Approximately 90% of gamers exhibit non-pathological use, with only 1–10% classified as addicted [32, 33]. Despite the fact that at present there is no unanimous opinion regarding the classification of high-calorie palatable foods as a factor of chemical or behavioral addiction [12, 13], the authors consider it justified to separate animals consuming chocolate into those who are prone or not prone to overeating it. However, given the heterogeneity of the data, an expansion of the sample and a set of behavioral tests is required to identify a clear separation criterion.

Agrelax has previously been reported to reduce compulsivity, anxiety, and exploratory behavior in rats [30, 34]. Danio rerio has also been shown to reduce kinase activity primarily in the forebrain and ghrelin and orexin levels in all brain regions after incubation with agrelax [35, 36]. With regard to the effect of agrelax on high-calorie food intake, its high efficacy in reducing chocolate treat intake has been documented in the limited access model without stress exposure, as well as with the addition of foot shock, acute vital stress, and chronic maternal deprivation stress [30, 34, 37]. In addition, it has been described that agrelax reduces emotional overeating induced by activation of the lateral hypothalamic reward system [38].

According to the results of the present study, using the elevated plus maze and ball burying tests, it was shown that rats prone to overeating chocolate treat (more than 36 kcal in one hour) demonstrated significantly greater anxiety and compulsivity compared to those who did not abuse the treat, as well as to animals of the control group, which were never offered chocolate. At the same time, rats not prone to overeating chocolate treat generally had a similar level of anxiety and compulsivity to the control.

As in the behavioral test experiment, in the experiment investigating the effect of stress and agrelax on eating behavior, the number of rats consuming more or less than 36 kcal of chocolate treat was divided approximately equally. For the former, the stress of electrical stimulation of the limbs significantly increased the consumption of chocolate treat, while for rats not prone to overeating, the values did not differ from the control. Agrelax significantly decreased the consumption of chocolate treat, stimulated by the effect of stress, in rats prone to overeating to the values obtained in animals not exposed to stress. Interestingly, in rats not prone to overeating, agrelax, on the contrary, increased the amount of treat consumed compared to the control group, which requires further study.

Conclusion

The results of the present study indicate that the peptide antagonist of the ghrelin receptor agrelax, synthesized by the staff of the Department of Neuropharmacology, Institute of Experimental Medicine (Saint Petersburg, Russia), significantly reduces the consumption of chocolate treat increased by the effect of stressful electrical stimulation of the limbs in rats prone to overeating high-calorie foods.

Additional Info

Author contributions: M.A. Netesa, N.D. Nadbitov: investigation, formal analysis, writing—original draft, writing—review & editing; P.D. Shabanov, A.A. Lebedev: conceptualization. All the authors approved the version of the manuscript to be published and agreed to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval: The conduct of the study was approved by the local ethical committee of the Institute of Experimental Medicine (protocol No. 2/23 dated 2023 June 15).

Funding sources: The study was carried out within the framework of the state assignment of the Ministry of Education and Science of the Russian Federation FGWG-2025-0020 for 2025–2027. “Search for molecular targets for pharmacological action in addictive and neuroendocrine disorders with the aim of creating new pharmacologically active substances acting on CNS receptors”.

Disclosure of interests: The authors have no relationships, activities, or interests for the last three years related to for-profit or not-for-profit third parties whose interests may be affected by the content of the article.

Statement of originality: No previously obtained or published material (text, images, or data) was used in this study or article.

Data availability statement: All data obtained in this study are available in this article.

Generative AI: No generative artificial intelligence technologies were used to prepare this article.

Provenance and peer-review: This paper was submitted unsolicited and reviewed following the standard procedure. The peer review process involved one external reviewer, a member of the Editorial Board, and the in-house science editor.

Disclaimer: This article is published as submitted by the authors. The au­thors are solely responsible for the content and style of the manuscript.

Дополнительная информация

Вклад авторов. М.А. Нетеса, Н.Д. Надбитова — проведение исследования, анализ данных, написание черновика рукописи, пересмотр и редактирование рукописи; П.Д. Шабанов, А.А. Лебедев — определение концепции. Все авторы одобрили рукопись (версию для публикации), а также согласились нести ответственность за все аспекты настоящей работы, гарантируя надлежащее рассмотрение и решение вопросов, связанных с точностью и добросовестностью любой ее части.

Этическая экспертиза. Проведение исследования одобрено локальным этическим комитетом ФГБНУ «Институт экспериментальной медицины» (протокол № 2/23 от 15.06.2023).

Источники финансирования. Исследование выполнено в рамках государственного задания Минобрнауки России FGWG-2025-0020 на 2025–2027 гг. «Поиск молекулярных мишеней для фармакологического воздействия при аддиктивных и нейроэндокринных нарушениях с целью создания новых фармакологически активных веществ, действующих на рецепторы ЦНС».

Раскрытие интересов. Авторы заявляют об отсутствии отношений, деятельности и интересов за последние три года, связанных с третьими лицами (коммерческими и некоммерческими), интересы которых могут быть затронуты содержанием статьи.

Оригинальность. При создании настоящей работы авторы не использовали ранее опубликованные сведения (текст, иллюстрации, данные).

Доступ к данным. Все данные, полученные в настоящем исследовании, доступны в статье.

Генеративный искусственный интеллект. При создании настоящей статьи технологии генеративного искусственного интеллекта не использовали.

Рассмотрение и рецензирование. Настоящая работа подана в журнал в инициативном порядке и рассмотрена по обычной процедуре. В рецензировании участвовали один внешний рецензент, член редакционной коллегии и научный редактор издания.

Дисклеймер: Данная статья публикуется в том виде, в каком она была представлена авторами. Авторы несут полную ответственность за со­держание и стиль рукописи.

 

¹ Public organization “Russian Association of Endocrinologists”. Clinical guidelines for “Obesity” (In Russ.) [cited 21 May 2025]. Available from: https://www.endocrincentr.ru/sites/default/files/specialists/science/clinic-recomendations/ozhirenie_vzroslye.pdf

² American Addiction Centers. Alcohol and Drug Abuse Statistics (Facts About Addiction) [updated 2025 Mar 26; cited 2025 May 26]. Available from: https://americanaddictioncenters.org/rehab-guide/addiction-statistics-demographi

About the authors

Mariia A. Netesa

Institute of Experimental Medicine

Author for correspondence.
Email: saintula@gmail.com
ORCID iD: 0009-0002-7353-1745
SPIN-code: 8429-6486
Russian Federation, Saint Petersburg

Natalia D. Nadbitova

Institute of Experimental Medicine

Email: natali_805@mail.ru
ORCID iD: 0000-0002-2957-226X
SPIN-code: 4153-1270

MD, Cand. Sci. (Medicine)

Russian Federation, Saint Petersburg

Andrei A. Lebedev

Institute of Experimental Medicine

Email: aalebedev-iem@rambler.ru
ORCID iD: 0000-0003-0297-0425
SPIN-code: 4998-5204

Dr. Sci. (Biology), Professor

Russian Federation, Saint Petersburg

Petr D. Shabanov

Institute of Experimental Medicine

Email: pdshabanov@mail.ru
ORCID iD: 0000-0003-1464-1127
SPIN-code: 8974-7477

MD, Dr. Sci. (Medicine), Professor

Russian Federation, Saint Petersburg

References