Reduction of compulsive overeating in rats caused by maternal deprivation in early ontogenesis with the use of a new ghrelin receptor antagonist agrelax

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INTRODUCTION

Compulsive (binge) overeating disorder is characterized by the intermittent and excessive consumption of palatable food in short periods. Unlike bulimia or anorexia nervosa, this behavior is not accompanied by compensatory behaviors [1]. Factors triggering episodes of compulsive overeating include mental and physical stress and dietary restrictions on high-calorie foods.

Intermittent consumption of high-calorie foods was shown in a rodent model to cause binge overeating, irrespective of weight gain or obesity [2]. Several rodent models of binge overeating were investigated. Intermittent exposure to a fat source was used in a restricted access model to induce episodes of overeating [2]. The experiment indicated the involvement of several neurotransmitter systems in binge overeating, particularly opioids, serotonin, dopamine, and hormones [3].

Ghrelin, an orexigenic peptide identified and described in the late 20th century, comprises 28 amino acids. Ghrelin is predominantly synthesized in the lateral region of the arcuate complex of hypothalamic nuclei by endocrinocytes of the cardiac mucosa of the cardiac part of the stomach and enters the bloodstream [4]. Ghrelin isoforms include acylated ghrelin (acetyl-ghrelin), nonacylated (desacyl-ghrelin), and obestatin, as well as two molecular forms of the ghrelin receptor (GHSR1A and GHSR1B). GHSR1A is the only one with biological activity [4]. Ghrelin receptors are localized in the digestive tract, adrenal glands, thyroid gland, heart muscle, and brain (hypothalamus, pituitary, hippocampus, amygdala, trunk nuclei, and neocortex) [4]. Ghrelin regulates eating behavior, psychostimulant and alcohol dependency [5], and stressor reactions [6]. Patients with compulsive overeating had decreased levels of peripheral ghrelin before eating, possibly related to the disorder’s neurochemical mechanisms [7].

There is currently a lack of research papers addressing the involvement of the ghrelin system in food addiction. The mechanisms of ghrelin receptors’ impact on reinforcement and emotional behavior systems under various environmental influences are unclear. Agrelax, a peptide antagonist of ghrelin developed by the Institute of Experimental Medicine, is active against GHSR1A ghrelin receptors [8].

The study aimed to investigate the effect of a new antagonist of ghrelin receptors agrelax on compulsive overeating induced by maternal deprivation in early ontogenesis in adult rats.

MATERIALS AND METHODS

The experiments were conducted on 32 male and five female Wistar rats weighing 200–250 g from the Rappolovo Laboratory Animal Nursery (Leningrad region). The animals were kept in vivarium conditions in standard plastic cages with free access to water and food from 8:00 to 20:00 at 22°C ± 2°C under inverted light conditions. During the experiment, the principles of humane laboratory rat treatment were observed in accordance with the Rules of Laboratory Practice in the Russian Federation (Order of the Ministry of Health of the Russian Federation, 2003, No. 267).

After they arrived from the nursery, the animals were quarantined for 2 weeks in the appropriate block of the vivarium. Female Wistar rats were kept in plastic cages (40 × 50 × 20 cm) of five individuals, each with access to water and food ad libitum. One male was placed in each cage. The next day, females’ vaginal swabs were obtained to identify spermatozoa, and the onset of pregnancy was recorded using light microscopy, which was considered Day 0. Animals were housed in separate cages after pregnancy onset. The pregnancy lasted 20 ± 2 days.

The animals were divided into groups. Group 1 included nonstressed animals who had access to the chocolate diet daily. Group 1md included animals after maternal deprivation accessing the chocolate diet daily. Group 2 consisted of nonstressed animals that accessed the chocolate diet thrice weekly. Group 2md had animals after maternal deprivation accessing the chocolate diet thrice weekly.

A model of maternal weaning

From Days 2 to 12 of the postnatal period, rats were placed in individual plastic cups for 180 min every day for 10 days. Visual contact with the mother was excluded. Rats were grown in standard cages with five individuals in each cage after maternal deprivation and milk feeding. The experiment used males aged 90–100 days and weighed 200–250 g.

Method of compulsive overeating of high-calorie food

Animals in the experimental groups were given 1-h access to a high-carbohydrate diet (Nutella chocolate paste-based mix) every day (group 1md) or every third day (group 2md). The control animals consumed only standard pelleted rat food. The high-calorie food consisted of a paste made from Nutella chocolate paste (Ferrero, Alba, Turin, and Italy), ground rat pellet food (4RF18; Mucedola; Settimo Milanese), and water in the following ratio: 52% chocolate paste, 33% food pellets, and 15% water. The diet had a calorie value of 3.63 kcal/g. Standard pelleted rat food was placed inside a metal mesh container suspended from the front wall of the cage; it was taken from the cage to estimate food intake by weighing it. The Nutella paste mixture was poured into a cup; the handle of the cup was put into the metal wall of the cage. The chocolate paste feeder was positioned within 5 cm of the animals and in full visual contact 15 min before feeding. The cup with the chocolate paste was placed inside a container with a metal mesh suspended from the cage’s front wall. The animal was able to see and smell the paste. During this 15-min period, the rat performed repetitive movements of the forelegs, head, and trunk to access the paste but failed to reach it. This manipulation increased serum corticosterone levels [9]. The cup was placed in the cage after 15 min to make the paste available to the rats. Before the overeating period, the standard rodent food in each cage was weighed to estimate the following day’s 24-h food intake. The rats were situated in separate cages 15 days after the start of the chocolate diet experiment and fed the chocolate diet for another 30 days. The amount of standard food eaten and the amount of chocolate paste eaten per 1 h of access were recorded. The weight of the animals was measured once a week on a certain day.

In Week 6 of the experiment, rats were administered a peptide analog of the ghrelin antagonist agrelax intranasally at a dose of 1 μg/1 μL, 20 μL for 7 days, which was synthesized in the S.V. Anichkov Department of Neuropharmacology at the Institute of Experimental Medicine using a genetically engineered method [8].

The Graph Pad Prizm v.6 software was used for statistical processing of quantitative data. The statistical significance of differences between groups was determined using a one-factor analysis of variance. Only two groups were compared using the Student’s t test for independent samples.

RESULTS AND DISCUSSION

When the effect of maternal deprivation on daily chocolate intake was examined, it was shown that the average daily chocolate consumption over 10 days of testing decreased (p < 0.05) in group 1md compared with the control (intact) group 1. Similarly, it reduced (p < 0.05) when aggrelax was introduced compared with the control (intact) group 1. When the effect of maternal deprivation on standard feed intake was investigated, the average daily intake in group 1md was found to be unchanged relative to the control (intact) group 1 and did not differ from the control (intact) group 1 after the introduction of aggrelax (Fig. 1).

 

Fig. 1. The effect of maternal deprivation on the consumption of standard feed and chocolate during daily chocolate delivery. *p < 0.05 relative to the control (intact) group of animals

Рис. 1. Влияние материнской депривации (МД) на потребление стандартного корма и шоколада при ежедневной выдаче шоколада. *p < 0,05 относительно контрольной (интактной) группы животных

When the effect of maternal deprivation on chocolate consumption was studied three times a week, it was found that the average daily chocolate intake over the 10 days of testing was increased (p < 0.001) in group 2md relative to the control (intact) group 2 and remained unchanged relative to the control (intact) group 2 after consumption of the standard feed. The average daily intake of chocolate in group 2md after administration of aggrelax three times per week for 10 days did not differ relative to the control (intact) group 2 and did not differ from the control (intact) group 2 after consumption of standard feed (Fig. 2).

 

Fig. 2. The effect of maternal deprivation on the consumption of standard feed and chocolate paste when giving out chocolate 3 times a week. The average daily intake is shown. **p < 0.001 relative to the control (intact) group of animals

Рис. 2. Влияние материнской депривации (МД) на потребление стандартного корма и шоколадной пасты при выдаче шоколада 3 раза в неделю. Показано среднее суточное потребление. **p < 0,001 относительно контрольной (интактной) группы животных

 

The term “food addiction” refers to compulsive eating behavior associated with a lack of control over food [1, 2]. Eating behavior can be regulated by both homeostatic (related to energy need/storage) and hedonic (dopaminergic brain reward system) pathways, which control energy intake and body weight [10].

The present study produced compulsive overeating using the high-calorie food overeating method. Chronic maternal deprivation stress was shown to cause increased signs of compulsive overeating of high-calorie foods. In a restricted access model of compulsive overeating, episodes of overeating are generated by intermittent exposure to a carbohydrate and fat source [2]. Our findings of reduced high-calorie food intake in rats after maternal deprivation with a daily food ration support using an intermittent food regimen in developing compulsive overeating. In the control group of rats who received chocolate paste daily, elements of compulsive overeating behavior and withdrawal from the chocolate diet were found. This can be explained by the inclusion in the experiment of elements of waiting for food for 15 min during visual contact with food, which is consistent with literature data [9].

The chronic stress of maternal deprivation in animals is a model of human maternal neglect. Data from an experimental model of weaning from the mother in early ontogenesis show that stress significantly impacts the formation of compulsive overeating [9]. Early mental stress has a long-term impact on development and socialization in children and adolescents, increasing the risk of developing eating disorders and compulsive overeating. Adolescence is characterized by hormonal changes and an imbalance of excitation and inhibition processes, highlighting the vital role of neurochemical intracerebral processes in the formation of compulsive overeating [9].

Experimental modeling of several clinical manifestations allows direct investigation of neurochemical mechanisms underlying compulsive overeating. The experiment demonstrated the involvement of neuroendocrine processes and several neurotransmitter systems, particularly in its occurrence. In addition, opioid, dopamine, and serotonin systems are involved in forming positive emotions in compulsive overeating [3]. In addition to these primary mediators, the ghrelin system is involved in the mechanisms of compulsive overeating, as demonstrated in the present study.

Ghrelin acts primarily in the hypothalamus and stimulates eating behavior to regulate energy homeostasis [11]. Previously, we found that chronic stress of isolation rearing activated eating behavior and weight gain in rats [12]. Ghrelin signaling in brain regions outside the hypothalamus is important because of its impact on learning and memory, reward and motivation, and anxiety and depression. Corticoliberin-producing (CRH) neurons of the hypothalamic paraventricular nucleus are potential targets of ghrelin activity during stress. Ghrelin administration has been demonstrated to activate these neurons [13]. The target of ghrelin action under stress appears to be the extended amygdala system, which includes the bed nucleus of the terminal striatum, the central nucleus of the amygdala, the nerveless substance, and the shell of the contiguous nucleus, being an extrahypothalamic CRH system [13]. The extended amygdala structures receive inputs from dopaminergic neurons in the ventral tegmental area and are the main functional system for realizing the emotional–motivational effects of various narcogens. Blocking CRH in the central nucleus of the amygdala, the bed nucleus of the terminal striatum, and the contiguous nucleus eliminates or significantly reduces the activating effects of addictive drugs [14].

CONCLUSIONS

In conclusion, the study found that chronic maternal deprivation stress causes increased signs of compulsive overeating of high-calorie foods. Intranasal administration of agrelax, a novel ghrelin receptor antagonist, reduces food addiction manifestations in rats after maternal deprivation with intermittent consumption of high-calorie foods. This approach suggests new methods for synthesizing and using peptide-based pharmacological agents based on ghrelin and its antagonists for treating food addiction caused by psychogenic stresses in early ontogenesis. When daily chocolate consumption was examined, the average daily consumption, pre- and postinduction of agrelax, was lower (p < 0.005) in the maternal deprivation group than in the control group, indicating no food addiction was evident. Therefore, the development of compulsive overeating in rats with maternal deprivation depends on intermittent consumption of high-calorie tasty foods.

ADDITIONAL INFORMATION

Authors’ contribution. Thereby, all authors made a substantial contribution to the conception of the study, acquisition, analysis, interpretation of data for the work, drafting and revising the article, final approval of the version to be published and agree to be accountable for all aspects of the study. The contribution of each author: A.A. Lebedev, S.S. Pyurveev, N.D. Nadbitova, A.V. Lizunov, E.R. Bychkov, V.V. Lukashkova, N.R. Evdokimova, M.A. Netesa, V.A. Lebedev — writing an article, data analysis; P.D. Shabanov — editing an article, developing a general concept.

Competing interests. The authors declare that they have no competing interests.

Funding source. The work was carried out within the framework of the state task of the Ministry of Education and Science of Russia FGWG-2022-0004 for 2022–2025 “Search of molecular targets for pharmacological action in addictive and neuroendocrine disorders and the creation of new pharmacologically active substances acting on CNS receptors”.

About the authors

Andrey А. Lebedev

Institute of Experimental Medicine

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

Dr. Biol. Sci. (Pharmacology), professor, head of the Laboratory of General Pharmacology, Department of Neuropharmacology

Russian Federation, Saint Petersburg

Sarng S. Pyurveev

Institute of Experimental Medicine; Saint Petersburg State Pediatric Medical University

Email: dr.purveev@gmail.com
ORCID iD: 0000-0002-4467-2269
SPIN-code: 5915-9767

junior research associate, Department of Neuropharmacology; assistant professor

Russian Federation, Saint Petersburg; Saint Petersburg

Natalia D. Nadbitova

Institute of Experimental Medicine

Email: natali_805@mail.ru

Cand. Sci. (Med.), research associate, Department of Neuropharmacology

Russian Federation, Saint Petersburg

Aleksey V. Lizunov

Institute of Experimental Medicine

Email: izya12005@yandex.ru

Aleksey V. Lizunov, Cand. Sci. (Biol.), research associate, Department of Neuropharmacology

Russian Federation, Saint Petersburg

Eugenii R. Bychkov

Institute of Experimental Medicine

Email: bychkov@mail.ru
ORCID iD: 0000-0002-8911-6805
SPIN-code: 9408-0799

Cand. Med. Sci. (Pathophysiology), head of the Laboratory, Department of Neuropharmacology

Russian Federation, Saint Petersburg

Valeriya V. Lukashova

Institute of Experimental Medicine

Email: aalebedev-iem@rambler.ru

postgraduare student, Department of Neuropharmacology

Russian Federation, Saint Petersburg

Natalia R. Evdokimova

Institute of Experimental Medicine

Email: aalebedev-iem@rambler.ru

Cand. Sci. (Biol.), research associate, Department of Neuropharmacology

Russian Federation, Saint Petersburg

Maria A. Netesa

Institute of Experimental Medicine

Email: saintula@gmail.com

postgraduate student, Department of Neuropharmacology

Russian Federation, Saint Petersburg

Viktor A. Lebedev

Institute of Experimental Medicine

Email: vitya-lebedev-57@mail.ru
ORCID iD: 0000-0002-1525-8106
SPIN-code: 1878-8392

Cand. Sci. (Biol.), research associate, Department of Neuropharmacology

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

Dr. Med. Sci. (Pharmacology), professor and head of the Department, Department of Neuropharmacology

Russian Federation, Saint Petersburg

References