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. 2024 Feb 1:15:1281135.
doi: 10.3389/fendo.2024.1281135. eCollection 2024.

Influence of leptin and its receptors on individuals under chronic social stress behavior

Renata M F Mélo  1 , Rafaela S Barbosa  1 , Victória L Ozório  1 , Gabriel M Oliveira  2 , Samuel I M Horita  1   3 , Andrea Henriques-Pons  1 , Tânia C Araújo-Jorge  1 , Viviane M S Fragoso  1
Affiliations

Influence of leptin and its receptors on individuals under chronic social stress behavior

Renata M F Mélo et al. Front Endocrinol (Lausanne). .

Abstract

Stress is the body's physiological reaction to a dangerous or threatening situation, leading to a state of alertness. This reaction is necessary for developing an effective adaptive response to stress and maintaining the body's homeostasis. Chronic stress, caused mainly by social stress, is what primarily affects the world's population. In the last decades, the emergence of psychological disorders in humans has become more frequent, and one of the symptoms that can be observed is aggressiveness. In the brain, stress can cause neuronal circuit alterations related to the action of hormones in the central nervous system. Leptin, for example, is a hormone capable of acting in brain regions and neuronal circuits important for behavioral and emotional regulation. This study investigated the correlation between chronic social stress, neuroendocrine disorders, and individual behavioral changes. Then, leptin and its receptors' anatomical distribution were evaluated in the brains of mice subjected to a protocol of chronic social stress. The model of spontaneous aggression (MSA) is based on grouping young mice and posterior regrouping of the same animals as adults. According to the regrouping social stress, we categorized the mice into i) harmonic, ii) attacked, and iii) aggressive animals. For leptin hormone evaluation, we quantified plasma and brain concentrations by ELISA and evaluated its receptor and isoform expression by western blotting. Moreover, we verified whether stress or changes in leptin levels interfered with the animal's body weight. Only attacked animals showed reduced plasma leptin concentration and weight gain, besides a higher expression of the high-molecular-weight leptin receptor in the amygdala and the low-molecular-weight receptor in the hippocampal region. Aggressive animals showed a reduction in the cerebral concentration of leptin in the hippocampus and a reduced high-and low-molecular-weight leptin receptor expression in the amygdala. The harmonic animals showed a reduction in the cerebral concentration of leptin in the pituitary and a reduced expression of the high-molecular-weight leptin receptor in the amygdala. We then suggest that leptin and its receptors' expression in plasma and specific brain areas are involved in how individuals react in stressful situations, such as regrouping stress in MSA.

Keywords: central nervous system; chronic stress; leptin; leptin receptor (LEPR); model of spontaneous aggression.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Experimental design of the model of spontaneous aggression (MSA) The male Swiss Webster mice were divided into five groups at the 3rd week of life (n=5 in each group), and behavioral tests, including the tail suspension test (TST), ethogram and pattern of aggressive behavior (PAB) test, were performed at the 4th, 6th and 8th weeks of life. At the 10th week of life, according to their mobility in the TST, the mice were classified as hyperactive (High), hypoactive (Low) or medium mobility (Med) and selected for regrouping. Most of the animals were regrouped into five groups with one High animal, one Low animal and three Med animals in each group. Regrouping represents a situation of social stress for animals. One group of 5 animals was not regrouped (NR), they were the social stress negative control. In the 12th, 14th and 16th weeks of life, we performed the aggressive ethogram and the PAB test. In the 16th week of life, the regrouped animals were categorized as harmonic (Har; social stress positive control), defeated (AgD) or highly aggressive (AgR). Then, the blood samples and cerebral regions were collected for ELISA and Western blot analysis.
Figure 2
Figure 2
Evaluation of behavioral parameters in social stress. (A) Distribution of animals according to the mobility profile obtained by the TST for regrouping in the 10th week of life. The groups were hyperactive (31,06%, dark blue), medium (44,10%, blue), and hypoactive (24,84%, light blue). (B) Number of attacks/30 min after regrouping, according to the categorization of animals into not regrouped (NR, gray circle), harmonic (Har, dark blue square), defeated (AgD, green triangle) and aggressive (AgR, inverted red triangle) groups. (C) Lesion extent (cm2) after regrouping according to the behavioral categories of NR, Har, AgD and AgR animals. (D) Evaluation of the pattern of aggressive behavior (PAB) after regrouping according to the behavioral categories of NR, Har, AgD and AgR animals. The PAB test was performed through an individual physical inspection of all animals, enabling the quantification of aggression according to predetermined scores: 0, no bites or injuries on the body; 1, reduced aggressive nonsexual events and discrete bite signs on the back or tail; 2, aggressive events and moderate lesions on the tail, back and scrotum; and 3, severe events of aggression and marked lesions on the tail, back and scrotum. The values are expressed according to the individual results of the mice from three independent experiments (NR, n= 15; Har, n= 20; AgD, n= 15; AgR, n=15 animals). **** corresponds p < 0.0001 between AgR and the other categories and AgD and the other categories by Kruskal-Wallis and Dunn's tests.
Figure 3
Figure 3
Evaluation of animal body weight under social stress. (A) Body weight of animals after being subjected to the social stress of regrouping according to the categorization of animals into not regrouped (NR, gray circle), harmonic (Har, dark blue square), defeated (AgD, green triangle) and aggressive (AgR, inverted red triangle) groups. (B) Variation in body weight of NR, Har, AgD, and AgR animals, according to the body weight change of each animal before (4th, 6th, and 8th weeks of age) and after (12th, 14th and 16th weeks of age) regrouping. The values are expressed according to the individual results of the mice from two independent experiments (NR, n= 10; Har, n= 12; AgD, n= 19; AgR, n=16 animals). * corresponds to p < 0.05 between AgD and NR, and #corresponds to p < 0.1 between AgD and Har by Kruskal-Wallis and Dunn's tests.
Figure 4
Figure 4
Evaluation of plasma leptin concentrations after social stress. Plasma leptin concentrations in not regrouped (NR, gray circle), harmonic (Har, dark blue square), defeated (AgD, green triangle) and aggressive (AgR, inverted red triangle) animals subjected to social regrouping stress. The values are expressed according to the individual results of the mice from two independent experiments (NR, n= 10; Har, n= 10; AgD, n= 13; AgR, n=14 animals). ** corresponds to p < 0.01 between AgD and NR. **** corresponds to p < 0.0001 between AgD and Har by Kruskal-Wallis and Dunn's tests.
Figure 5
Figure 5
Evaluation of the levels of cerebral leptin. Quantitative analysis of cerebral leptin expression by ELISA according to the categories NR (gray bar), Har (blue bar), AgD (green bar) and AgR (red bar) in the (A) pituitary gland, (B) hypothalamus, (C) hippocampus, (D) amygdala, and (E) prefrontal cortex. The values are expressed according to the individual results of the mice from two independent experiments (NR, n= 10; Har, n= 10; AgD, n= 13; AgR, n=14 animals). * corresponds to p < 0.05 between Har and NR, and AgR and NR by Kruskal-Wallis and Dunn's tests.
Figure 6
Figure 6
Expression levels of the high-molecular-weight leptin receptor. Quantitative analysis of high-molecular-weight leptin receptor expression by Western blotting analysis according to the categories NR (gray bar), Har (blue bar), AgD (green bar) and AgR (red bar) in the (A) hypothalamus, (B) hippocampus, (C) amygdala, and (D) prefrontal cortex. GAPDH was used as an internal control for the samples. The values are expressed as the variation index (V.I.) of the means ± standard deviations (SDs) of the analyzed samples from three independent experiments using the Kruskal-Wallis and Dunn's test (NR, n=9; Har, n=10; AgD, n=15; AgR, n=10 animals). * corresponds to statistical significance (p < 0.05).
Figure 7
Figure 7
Expression levels of the low-molecular-weight leptin receptor. Quantitative analysis of low-molecular-weight (~100 kDa) leptin receptor expression by Western blotting analysis according to the categories NR (gray bar), Har (blue bar), AgD (green bar) and AgR (red bar) in the (A) hypothalamus, (B) hippocampus, (C) amygdala, and (D) prefrontal cortex. GAPDH was used as an internal control for the samples. The values are expressed as the variation index (V.I.) of the means ± standard deviations (SDs) of the analyzed samples from three independent experiments using the Kruskal-Wallis and Dunn's test (NR, n=9; Har, n=10; AgD, n=15; AgR, n=10 animals). * corresponds to p < 0.05 between AgR and NR; ** corresponds to p < 0.01 between AgR and NR.
Figure 8
Figure 8
Analysis of leptin receptor expression levels according to molecular weight. Comparing leptin receptor expression levels of high (dark pink bars) and low molecular weight (light pink bars), according to the categories of NR, Har, AgD, and AgR animals, in the (A) hypothalamus, (B) hippocampus, (C) amygdala, and (D) prefrontal cortex. The values are expressed according to the indivi- dual results of the mice from three independent experiments. ** corresponds to p <0.01, and # corresponds to p <0.1 by Kruskal-Wallis and Dunn's tests.
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The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (BR)- Brazil (CNPq) and Instituto Oswaldo Cruz/Fundação Oswaldo Cruz (ioc-023-fio-18-2-52). The funders had no role in the study design, data collection, and analysis, decision to publish, or manuscript preparation.
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