Introduction: Given the suggested metabolic regulatory effects of stress-responsive genes and based on the impacts of early-life stress on HPA axis development, this study aimed to characterize the maternal separation (MS) impact on the communication between glucose metabolism and HPA axis dysregulations under chronic social defeat stress (CSDS). Methods: During the first 2 weeks of life, male Wistar rats were either exposed to MS or left undisturbed with their mothers (Std). Starting on postnatal day 50, the animals of each group were either left undisturbed in the standard group housing (Con) or underwent CSDS for 3 weeks. There were four groups (n = 10/group): Std-Con, MS-Con, Std-CSDS, and MS-CSDS. Results: Early and/or adult life adversity reduced β-cell number, muscular FK506-binding protein 51 (FKBP51) content, and BMI in adulthood. The reduction of β-cell number and BMI in the MS-CSDS rats were more profound than MS-Con group. CSDS either alone or in combination with MS reduced locomotor activity and increased and decreased corticotropin-releasing factor type 1 receptor (CRFR1) content, respectively, in hypothalamus and pancreas. Although, under CSDS, MS intensified HPA axis overactivity and reduced isolated islets’ insulin secretion, it could promote resilience to depression symptoms. No differences were observed in hypothalamic Fkbp5 gene DNA methylation and glucose tolerance among groups. Conclusion: MS exacerbated HPA axis overactivity and the endocrine pancreas dysfunctions under CSDS. The intensified corticosterone secretion and the diminished content of pancreatic CRFR1 protein could be involved in the reduced β-cell number and islets’ insulin secretion under CSDS. The decreased muscular FKBP51 content might be a homeostatic response to slow down insulin resistance development under chronic stress.

Environmental factors, particularly psychological stress-related circumstances or mental health problems (depression), have emerged as critical contributors to the development of metabolic disturbances [1]. Substantial evidence suggests that early-life programming of hypothalamus-pituitary-adrenal (HPA) axis reactivity is a crucial factor for psychological or metabolic disease susceptibility under chronic exposure to stressors later in life [2, 3]. In the early postnatal development, neonatal rodents indicate a weakened HPA axis reactivity to mild stressors, known as the stress hyporesponsive period. However, disruption of mother-offspring relationship, as an intense stressor, could effectively activate HPA axis and expose the developing organism to excessive circulating corticosterone (CORT) levels [4]. In this association, maternal separation (MS) as the most common experimental stress model in the early life could disturb stress hyporesponsive period and exert persistent alterations of neuroendocrine responses to stressful events in adulthood [5]. Since the main source of stressful events in humans is social stress, chronic social defeat stress using the resident-intruder paradigm as a familiar situation in the natural life of rodents could be reliable to model this situation in adult rats [6]. It has been reported that the elevated levels of circulating CORT impair insulin secretion from the islets of Langerhans and the insulin signaling cascade [7, 8], which are regarded as the major causes of glucose metabolism disorders, such as diabetes [9]. The secretion of stress hormones including CORT is regulated through a negative feedback loop, activated by CORT binding to the glucocorticoid receptor (GR) [10]. GR sensitivity is mainly regulated by its bound chaperones, specifically the heat shock protein 90 and its co-chaperone FK506-binding protein 51 (FKBP51) (for review, see [11]). Chronic stress or glucocorticoid-induced upregulation of FKBP51 limits sensitivity and in turn the nuclear translocation of GR via an ultra-short negative feedback loop, causing prolonged CORT secretion [11]. This turns the Fkbp5 gene into a main candidate for gene-environment cross-talk, affecting an individual’s sensitivity to the pathogenesis of stress-related disorders [12]. Epigenetic changes, especially modifications in DNA methylation profiles, can mediate the effects of genome-environmental interactions, such as early-life stress (ELS) into the expression changes of key-regulatory genes of the HPA axis [13‒15], among which the Fkbp5 gene is mostly recommended [16]. Furthermore, hypothalamic corticotropin-releasing factor type 1 receptor (CRFR1) is another important central component of HPA axis regulation that is positively regulated by glucocorticoids and employed specifically during chronic stressful conditions [17]. In addition, FKBP51 and CRFR1 are strongly suggested to mediate the cross-talk between stress and metabolic systems owing to the high expression in metabolically relevant tissues in the periphery [18] and β cells of pancreatic islets [19, 20], respectively. Accordingly, it has been recognized that Fkbp5 deletion or treatment with pharmacological antagonism of FKBP51 in mice markedly lowered fasting glucose and improved glucose tolerance through GLUT4 recruitment to the skeletal muscle membrane, under high fat diet conditions [18]. CRFR1 has also been recognized to express in the pancreatic β cells by increasing glucose-stimulated insulin secretion (GSIS) and β-cell proliferation [19, 21]. So, it might be contributed to the communication between HPA axis and the endocrine pancreas function under chronic exposure to stress [20, 22]. To the best of our knowledge, despite the suggested regulatory role of FKBP51 and CRFR1 in glucose metabolism, the alterations of these molecules in peripheral metabolically active tissues under early and/or adult life adversity have not yet been investigated. Regarding this, we explored whether MS could intensify glucose metabolism alterations, including pancreatic islets’ insulin secretion and content, β-cell number, and glucose tolerance in response to chronic social defeat stress (CSDS) in adulthood. Furthermore, to characterize the suggested regulatory role of CRFR1 and FKBP51, as key regulators of the stress hormone system, respectively, in insulin secretion and glucose tolerance, pancreatic CRFR1 and skeletal muscle-specific FKBP51 contents along with their hypothalamic changes were measured. We focused on the skeletal muscle since it accounts for almost 80% of postprandial glucose disposal and is the main site responsible for keeping glucose homeostasis. Furthermore, in this study the regional methylation differences of CpG islands within the promoter of Fkbp5 gene in the hypothalamus were investigated. The behavioral tests (open field, sucrose preference, and social avoidance) were also performed to confirm our stress paradigm validity. We hypothesized that (1) MS would intensify the basal plasma CORT levels and depression symptoms in response to CSDS in adulthood; (2) changes in the CORT levels would go along with changes in the hypothalamic protein contents of FKBP51 and CRFR1 and regional DNA methylation patterns of the hypothalamic Fkbp5 gene promoter; and (3) increase of FKBP51 and decrease of CRFR1 protein contents, respectively, in soleus muscle and pancreas, could be followed by the development of insulin resistance and reduction of β-cell number and insulin secretion capacity under chronic stressful conditions.

Animals

Ten female and five male Wistar rats (200–250 g) purchased from the Pasture Institute (Tehran, Iran) were mated overnight and separated the following morning. The pregnant rats were kept in a temperature-controlled room (22°C ± 2°C) with a 12-h light/dark cycle (lights turned on at 0700) and their food and water were provided ad libitum. The birth day was termed postnatal day 0 (PND 0). The litter size was about 8–12 pups, 3–6 were male. On PND 1, concerning the limited current knowledge on the MS mechanisms and the probable interaction of the female sex hormones with the stress hormones, the female pups were removed [23]. However, the findings can be utilized as a basis for future research on female rats. Notably, the litter size was adjusted to 3–6 male pups. According to Champagne et al. [24], neither the size nor gender composition of the litter significantly influences the frequency of maternal licking/grooming, or arched-back nursing and, thus, has little effect on HPA axis development or central expression levels of stress-related genes [25]. Furthermore, it has been demonstrated that rats raised in small litters of 2 or 4 exhibited no significant differences in body weight or composition from rats raised in litters of 8 or 12 [26]. The pups of ten litters were randomly divided into the two following groups (20 rats/group, five litters/group): standard rearing environment (Std) and MS groups. At puberty (PND 50), the animals of each group were then randomly divided into two subgroups (10 rats/group, five litters/group): control (Con) and CSDS subgroups. A total of 40 male offspring were used in the present study. One or two offspring from each litter were used in each biochemical or molecular experiment to avoid litter bias.

Experimental Design

On PND 1, the litters were randomly divided into two groups (five litters/group): standard rearing environment (Std) and MS groups. MS was conducted from PND 1 to PND 14 for 3 h per day at random times between 0800 and 1,800 [5]. Separated pups were moved to an adjacent room and kept apart from their siblings in small cages with clean sawdust while the mothers were left undisturbed. Standard reared litters (Std group) were left undisturbed. Pups were weaned on PND 21 and placed in groups of two to three per cage according to postnatal exposure to MS or no stress (Std) and left undisturbed until puberty (20 rats/group, five litters/group). Starting on PND 50, the animals of each group were either left undisturbed in standard housing conditions (Con) or underwent CSDS for 3 weeks. The animals of Std-CSDS and MS-CSDS subgroups were daily replaced with companion-sterilized females in the home cage of the unfamiliar aggressive male residents (300 ± 30 g). Thus, there were a total of four groups (10 rats/group, five litters/group): standard reared control rats (Std-Con), maternal separated rats (MS-Con), rats undergoing chronic social defeat stress (Std-CSDS), and those undergoing both maternal separation and chronic social defeat stress (MS-CSDS). All the animals underwent locomotor activity (open field) and depression behavior (sucrose preference and social avoidance) assessing tests in the last week of the experiment. Body weight was measured daily for the first 14 days and then weekly from PND 15 to the end of the study. Body length and food intake were recorded weekly from PND 1 and PND 23, respectively, onward. Lee index and body mass index (BMI) were calculated as follows:

graphic

graphic

Plasma CORT levels were measured on PNDs 22, 51, 66, and 72 and an oral glucose tolerance test (OGTT) was performed on PND 70. GSIS and content of isolated pancreatic islets were measured at two different glucose concentrations (5.6 and 16.7 mM). Moreover, Adrenal, thymus, and intra-abdominal fat were removed and weighted quickly (Fig. 1). Tissue contents of FKBP51 (in hypothalamus and soleus muscle) and CRFR1 (in hypothalamus and pancreas) proteins along with regional CpG methylation status of the hypothalamic Fkbp5 gene promoter were assessed at the end of the research.

Fig. 1.

Experimental timeline. Std, standard rearing; Con, no adult stress; MS, maternal separation; CSDS, chronic social defeat stress (10 rats/group, five litters/group).

Fig. 1.

Experimental timeline. Std, standard rearing; Con, no adult stress; MS, maternal separation; CSDS, chronic social defeat stress (10 rats/group, five litters/group).

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Chronic Social Defeat Stress

The CSDS paradigm involves the daily introduction of the experimental rat (intruder) into the territory of a physically superior aggressive animal (resident) to be defeated for 3 weeks [27]. Briefly, an intruder was replaced a cohabitating sterilized female (by ligation of the oviducts) in the home cage of an unfamiliar, aggressive resident male rat (100 × 60 cm, h: 30 cm). In social defeat models using rats, aggression and territorial dominance of residents toward intruder males can be established by pair-housing with a ligatured female [28]. In this way, the female kept hormonally intact and will be commonly receptive without becoming pregnant and developing maternal aggression [29]. The intruders were defeated and forced into subordination including attacks, bites, and threats by the residents for 10 min after the first attack. Since social defat stress was the main aim of our study, highly aggressive phenotypes as residents were used. So, physical injury was inevitable, but it should not be mixed with the stress of severe physical harm. It is noteworthy that the physical injury in a nonviolent social defeat confrontation includes biting as brief nipping of the skin mainly at the back and flanks of the intruder with thick and tough skin, which leaves behind small imprints of the incisors and does not require any veterinary care [30]. Therefore, the confrontation should be terminated earlier upon the resident that shows signs of violence causing bite wounds at vulnerable body parts (belly, throat, and paws) and the violent resident should be excluded from the experiment [29]. Right after the physical defeat session, the intruders were separated from the residents with a perforated transparent partition which allowed psychogenic contacts to the resident without physical harm for 1 h. The daily defeat was performed during the dark phase (the main activity phase) with different resident rats to minimize repeated encounters throughout the experiment. Offensive aggression behaviors of the animals during the physical defeat session were manually recorded. In this study, the average time between introduction of the intruder and the first clinch attack was 52 s. The frequency of the lateral threat, upright posture, clinch, biting, and keep down was 2.57, 0.80, 2.88, 3.24, and 2.76, respectively. It is worth noting that 20 intact male resident Wistar rats (300 ± 30 g) bought from Pasture Institute (Tehran, Iran) were trained for aggressive behavior (attacks, bites, and threats) and were screened for choosing 10 of the most aggressive individuals before the onset of the actual start of the social stress experiment.

Blood Sampling and Tissue Collection

The overnight fasted (16–18 h) animals were briefly exposed to isoflurane (Nicholas Primal, London, UK) as an anesthetic agent and blood samples were taken by retro-orbital puncture method (0800–0830) [31]. For determination of basal plasmatic CORT levels, blood samples were collected in distinct moments: weaning (PND 21), 1 day after the first defeat (PND 51), during social defeat stress (PND 66), and 1 day after the last defeat (PND 72). All the blood samples were collected into heparinized plastic tubes (5,000 IU/mL) (10 μL/mL) and were subsequently centrifuged (10 min at ×664 g). The separated plasma was stored at −80°C until assay. On PND 72, anesthetized rats (with isoflurane inhalation) were decapitated and hypothalamus, soleus muscle, and pancreas (5 rats/group, five litters/group) were dissected out and transferred to liquid nitrogen quickly and stored at −80°C. Following the Paxinos atlas [32], the brain was placed in a position with the hypothalamus upward and the cerebral hemispheres downward in foil over dry ice. Hypothalamus samples were obtained using a sterilized stainless steel forceps [33]. For extraction of soleus muscle, the skin of the leg was first removed. Afterward, the Achilles tendon was cut as close as possible to the foot with scissors. The gap was opened up to the knee and the soleus muscle was recognized by its darker pink color. After the whole muscle was freed from fascia, a cut was made at the tendons. The samples were homogenized in the ice-cold lysis buffer (NaCl 150 mM, sodium deoxycholate 0.25%, Triton X-100 0.1%, Tris HCl 50 mM, SDS 0.1%, EDTA 1 mM, and protease inhibitor cocktail [Sigma]) and centrifuged at ×1,250 g for 30 min at 4°C. The supernatants were collected and stored at −80°C.

Protein Assay and Measurement of the Protein Content of FKBP51 and CRFR1

Total concentration of protein was estimated using Bradford method [34] and a standard curve was generated using bovine serum albumin as the standard. FKBP51 content of hypothalamus and soleus muscle was measured using a rat enzyme-linked immunosorbent assay (ELISA) (Cat. No.: PTC-16355-R9648, sensitivity: 1.87 pg/mL) (ZellBio, Germany) and the intra-assay coefficient of variations was 5.7%. CRFR1 content of hypothalamus and pancreas was measured using a rat ELISA kit (Cat. No.: PTC-16352-R9648, sensitivity: 0.039 ng/mL) (ZellBio, Germany) and the intra-assay coefficient of variations was 5.7%.

Methylation-Specific PCR

DNA methylation on cytosine residues of CpG islands, mainly in the promoter region and first exon of many genes, is implicated in repressing the expression of downstream genes [35]. A methylation-specific PCR technique can be attempted to assess the methylation status of a limited number of CpG sites and simple yes/no results [36]. Regarding the Fkbp5 gene, the selected amplicon was an 80 bp sequence located before the exon 1 region. The criterion for choosing the target amplicons was based on the presence of CpG islands in the promoter region, preferably close to the transcription start site. This essay entails the initial sodium bisulfite treatment of genomic DNA, using an EpiTect Bisulfite Conversion kit (QIAGEN), converting all unmethylated cytosines to uracils but leaving the methylated cytosines unchanged, followed by subsequent amplification with two different primer pairs specific for either the methylated versus unmethylated DNA using PCR (consisted of 40 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, and final extension at 72°C for 7 min). Products were confirmed on agarose gels [37]. The following sets of primer pairs were designed using the Meth Primer software (http://www.urogene.org/methprimer/): The primer specific for methylated sequence forward: 5′ TAA GGG TTA GAT ACG TGG GTC-3′; reverse: 5′-GCA CTA TCT ATA CAA ATA AAC CTC CG-3′ and the primer specific for unmethylated sequence forward: 5′ TAA GGG TTA GAT ATG TGG GTT G-3′; reverse: 5′- ACA CTA TCT ATA CAA ATA AAC CTC CAC C-3′.

Biochemical Analyses of Plasma Parameters

Plasma levels of CORT were measured using a rat ELISA kit (Cat. No.: PTC-10496-R9648, sensitivity: 0.9 nmol/L) (ZellBio, Germany). The intra-assay coefficient of variations was 6.1%. Plasma glucose levels were measured using the glucose oxidase method (sensitivity: 1 mg/dL) (Parsazmun, Tehran, Iran). The intra-assay coefficient of variations was 2.1%. Plasma insulin concentration was measured using a rat insulin ELISA kit (Cat. No.: PTC-10707-R9648, sensitivity: 0.1 mIU/L) (ZellBio, Germany). The intra-assay coefficient of variations was 5.1%. Plasma leptin levels were assayed with ELISA diagnostic kit (Cat. No.: ZB-10561C-R9648, sensitivity: 0.05 ng/mL) (ZellBio, Germany).

Behavioral Assessments

Open-Field Test

Open-field test was performed in an open-field arena made of black Plexiglas (60 × 60 cm with 50 cm walls) on PND 68. The animals were placed in the center of a test chamber to freely explore for 10 min under low illumination (15 l×). The behavior of the animals was recorded with a camera above the maze hanging from the ceiling. Data were obtained using the behavioral tracking system Ethovision (Noldus, USA) and later analyzed for the total distance traveled reflecting locomotor activity [38]. The reduction of locomotor activity represents a loss of interest in new stimulating situations, implying a deficit in motivation [39]. Between each trial, the apparatus was completely cleaned with 10% ethanol.

Sucrose Preference Test

Sucrose preference test model is mainly conducted to dedicate anhedonia, a principal symptom of chronic depression that indicates loss of interest. The sucrose preference test was carried out as described elsewhere [38] with some modifications. Briefly, the rats were primarily habituated to consuming water in the two-bottles-choice paradigm for 3 days. On PND 67, the bottles were removed for 1 h to increase drinking behavior and then one of the bottles was replaced with 1% sucrose and the animals were allowed to choose between regular and sweet water for 12 h. At the beginning and end of the test, the bottles were weighed and consumption was calculated. Sucrose preference was calculated as a percentage of sucrose consumed over the total amount of liquid consumed:

graphic

Social Avoidance Test

The three-chamber apparatus was used to assess social avoidance at the end of the social defeat period (PND 71) [40]. In brief, the Plexiglas rectangular apparatus (57 × 23 × 31 cm) was partitioned into three equal chambers with transparent removable dividing walls. Inverted wire enclosure cups (diameter 12 cm) were placed in each side room. The test rat was first placed in the closed-off center chamber to acclimatize it for 5 min. A stranger male Wistar rat (8–10 weeks) was enclosed in a wire cup in either the left or the right chamber while the wire cup of opposite room was empty. The test rat was then allowed to explore for 10 min by removing the dividing walls. %social avoidance index was quantified using the following equation: %social avoidance index = (total duration of active contacts between the test rat with the empty enclosure cup/(total duration of active contacts between test rat with cup housing stranger + total duration of active contacts between test rat with the empty enclosure cup)) × 100.

The duration of active contacts was manually monitored and recorded by a blind observer at least 2 m away from the apparatus. The apparatus and wire cups were completely cleaned with 70% ethanol for each test rat.

Oral Glucose Tolerance Test

Oral glucose delivery during a GTT is a physiological route of glucose entry mainly because glucose absorption from the gut leads to the release of glucagon-like peptide 1 which in turn potentiates glucose-induced insulin release [41]. To perform the test, the overnight fasting (16–18 h) animals (PND 70) received a single 2 g/kg oral glucose (45% glucose solution) [42, 43] (Sigma), and blood samples were collected by retro-orbital puncture method immediately before and at 30, 60, and 120 min after glucose administration to measure plasma glucose and insulin concentrations [44]. Finally, the plasma parameters changes over time were linearly plotted and the area under curves (AUC) were calculated.

Calculation of Insulin Resistance and Sensitivity Indices and HOMA of β-Cell Function

Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as follows [45]:

graphic

Quantitative insulin sensitivity check index (QUICKI) [46] and ISI(Matsuda) [47] as insulin sensitivity indices were calculated as follows:

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graphic

HOMA of β-cell function (HOMA-β) was calculated as follows [47]:

graphic

Pancreatic Islet Isolation

The overnight fasting animals were decapitated on PND 72. Then, according to a method of Lacy and Kostianovsky [48], islets were isolated by injecting 10 mL ice-cold Hank’s balanced salt solution containing 0.5 mg/mL of collagenase P (Roche, Germany) via the common bile duct while the entrance of the duct to duodenum was clamped. The entire pancreas was then placed into a 50 mL falcon tube and incubated for 17 min at 37°C water bath. Digestion was terminated by adding ice-cold Hank’s balanced salt solution up to 40 mL and being shaken for 1 min. The digested suspension was placed into a glass container (7.5 cm diameter and 4.5 cm height) and washed thrice by adding cold Hank’s balanced salt solution and aspiration after precipitation. Islets were handpicked under a Blue Light stereomicroscope (USA) and used for insulin secretion and insulin content studies.

Glucose-Stimulated Insulin Secretion and Content of Pancreatic Isolated Islets

From the isolated islets of each of the noted animals (4 rats/group), two groups of ten islets were picked for each glucose concentration (second picking) and placed in plastic cups (8 cups in total for each condition). It is noteworthy that all earlier procedures were done on an ice tray. Then, the islets were incubated in 1 mL Krebs-Ringer solution (pH 7.4) containing (in mM) NaCl, 111; KCl, 5; CaCl2, 1; MgCl2 6H2O, 1; NaHCO3, 24 (Merck, Germany); HEPES, 10 (Sigma, USA); bovine serum albumin, 0.5 g/dL (Sigma, USA), and 5.6 or 16.7 mM glucose concentrations at 37°C water bath for 90 min (at the start, the cups were gassed with 95% O2/5% CO2 for 5 min). Then, the incubation medium was collected and stored at −80°C until the insulin measurement and the islets were incubated overnight at 4°C in 1 mL of acid ethanol (0.15 M HCl in 75% ethanol) to extract their insulin content [49]. The insulin secretion and content of islets were measured by a rat insulin ELISA kit (sensitivity: 0.1 mIU/L) (ZellBio, Germany). The intra-assay coefficient of variations was 5.1%.

Histological Analysis of the Pancreatic Islet Tissues

Gomora’s aldehyde fuchsin staining technique can produce intense staining of the insulin content of β cells. The chemical basis for the method was assumed to be the reaction of aldehyde fuchsin with insulin after prior oxidation by periodic acid [50]. One day after the last defeat (PND 72), the rats received an i.p. injection of mixed ketamine xylazine (100/20 mg/kg). Transcardial perfusion of 200 mL of cold 1% saline followed by 200 mL of 10% paraformaldehyde was carried out. The pancreas was taken from splenic regions and post-fixed in formalin overnight at 4°C, dehydrated by passing through a graded series of ethanol, and embedded in paraffin blocks. Then, 5 μm thick sections were prepared on a microtome. The sections were deparaffinized in xylene, rehydrated with serial dilutions of alcohol, and washed in distilled water. After embedding slides in aldehyde Fuchsin solution for 15 min, three exposures to 95% ethanol 3 min, 2 min, and 1 min were done, respectively. Then, slides were dipped in 70% ethanol, washed in tap water, and counterstained with Mayer’s hematoxylin and eosin. After washing in tap water, the sections were dehydrated, cleared in several changes of xylene, cover slipped, visualized under a light microscope Nikon E600 equipped with an optical camera, and were photographed at ×10 and ×40 magnification. Image J software was used to quantify the area of the islets and all nuclei of purple-violet stained cells inside the islet were counted as β-cell number. Twenty slides from the pancreas of each group were randomly selected and 4 islets were randomly examined on each slide. A total number of 80 islets of each group were counted.

Statistical Analysis

In the present study, results were presented as mean ± standard error of the mean (SEM) and were analyzed using GraphPad Prism version 8.0 and SPSS 21 software. Two-factor mixed-model ANOVA (considering time as the repeated factor and ELS as an independent factor), three-factor mixed-model ANOVA (considering time as the repeated factor and early and adult life adversity as independent factors), three-way ANOVA (considering glucose concentration, early and adult life adversity as independent factors), and two-way ANOVA (considering the independent factors of early and adult life adversity) followed by Tukey’s post hoc test were used. Unpaired t test was also used to compare serum CORT levels between standard reared and MS pups at weaning. Two-sided p values <0.05 were considered statically significant.

Contents of FKBP51 in Hypothalamus and Soleus Muscle and CRFR1 in Hypothalamus and Pancreas

Two-way analysis of variance followed by Tukey’s post hoc test for FKBP51 (early life adversity effect: F(1, 16) = 1.420, p = 0.251; adult life adversity effect: F(1, 16) = 42.00, p < 0.0001; early × adult life adversity: F(3, 16) = 3.336, p = 0.086; Table 1) and CRFR1 (early life adversity effect: F(1, 16) = 0.680, p = 0.421; adult life adversity effect: F(1, 16) = 24.70, p = 0.0001; early × adult life adversity: F(3, 16) = 0.2548, p = 0.6206; Table 1) contents in hypothalamus indicated a significant increase among Std-CSDS (p < 0.05) and MS-CSDS (p < 0.001, FKBP51; p < 0.05, CRFR1) rats compared to Std-Cons. On the other hand, FKBP51 content in soleus muscle was significantly decreased in response to either adversity during early life (MS-Con) (p < 0.01), adulthood (Std-CSDS) (p < 0.001), or both of them (MS-CSDS) (p < 0.05) compared to those under normal conditions (early life adversity effect: F(1, 16) = 2.647, p = 0.1233; adult life adversity effect: F(1, 16) = 10.90, p = 0.0045; early × adult life adversity: F(3, 16) = 13.19, p = 0.0022; Table 1). Similarly, pancreatic CRFR1 content was significantly reduced among Std-CSDS and MS-CSDS rats compared to Std-Cons (early life adversity effect: F (1, 16) = 1.106, p = 0.3087; adult life adversity effect: F(1, 16) = 26.25, p = 0.0001; early × adult life adversity: F(3, 16) = 1.378, p = 0.2576; Table 1) (p < 0.01). Statistical analysis also indicated a significant increase in hypothalamic FKBP51 (p < 0.001) and CRFR1 (p < 0.01) contents in MS-CSDS rats compared to MS-Con rats.

Table 1.

Protein contents of FKBP51 in hypothalamus and soleus muscle and CRFR1 in hypothalamus and pancreas (5 rats/group)

 Protein contents of FKBP51 in hypothalamus and soleus muscle and CRFR1 in hypothalamus and pancreas (5 rats/group)
 Protein contents of FKBP51 in hypothalamus and soleus muscle and CRFR1 in hypothalamus and pancreas (5 rats/group)

Methylation-Specific PCR

Fkbp5 genomic DNA sample yields only a positive signal in the methylation reaction and is a fully methylated sample in all groups. In other words, neither early nor adult life adversity affected methylation pattern of CpG islands located before the exon 1 region in 5′-end of Fkbp5 gene (Fig. 2b).

Fig. 2.

DNA methylation assessment: a schematic overview of promoter region of the Fkbp5 gene comprising sequence features such as CpG islands, GC percent, and CpG site. b Agarose gel electrophoresis demonstrating methylation status of targeted promoter region of Fkbp5 by methylation-specific PCR. Lane U and M amplified products with primers recognizing unmethylated and methylated Fkbp5 sequence of hypothalamus, respectively. Methylation-specific PCR products were separated on a 2.5% agarose gel containing ethidium bromide and visualized under UV illumination. Std-Con, rats under normal conditions; MS-Con, maternal separated rats; Std-CSDS, rats underwent chronic social defeat stress; MS-CSDS, rats underwent both maternal separation and chronic social defeat stress.

Fig. 2.

DNA methylation assessment: a schematic overview of promoter region of the Fkbp5 gene comprising sequence features such as CpG islands, GC percent, and CpG site. b Agarose gel electrophoresis demonstrating methylation status of targeted promoter region of Fkbp5 by methylation-specific PCR. Lane U and M amplified products with primers recognizing unmethylated and methylated Fkbp5 sequence of hypothalamus, respectively. Methylation-specific PCR products were separated on a 2.5% agarose gel containing ethidium bromide and visualized under UV illumination. Std-Con, rats under normal conditions; MS-Con, maternal separated rats; Std-CSDS, rats underwent chronic social defeat stress; MS-CSDS, rats underwent both maternal separation and chronic social defeat stress.

Close modal

Plasma CORT and Leptin Levels

Unpaired t test (t10 = 25.53, p = 0.026, Fig. 3a) demonstrated that MS pups had significantly higher plasma CORT levels than standard reared pups at weaning (PND 21) (p < 0.05). Three-factor mixed-model ANOVA followed by the Tukey’s test (time effect: F(2, 28) = 4.058, p = 0.025; early life adversity × time: F(1, 9) = 2.190, p = 0.125; adult life adversity × time F(1, 9) = 0.302, p = 0.741; early × adult life adversity × time: F(3, 28) = 5.194, p = 0.010; early life adversity effect: F(1, 5) = 48.531, p < 0.0001; adult life adversity effect: F(1, 5) = 6.268, p = 0.021; early × adult life adversity: F(3, 15) = 0.757, p = 0.395; Fig. 3b) showed a significant increase in CORT levels 1 day after the first defeat (PND 51; p < 0.01), during social defeat stress (PND 66; p < 0.001), and 1 day after the last defeat (PND 72; p < 0.05) in MS-CSDS rats compared to Std-Cons. Std-CSDS rats also presented increased CORT levels on PND 51 and PND 66 compared to Std-Cons (p < 0.05). On PNDs 66 and 72, a significant increase in CORT levels was detected in MS-CSDS rats compared to MS-Cons (p < 0.001). The calculated AUC confirmed the mentioned increase of this parameter (early life adversity effect: F(1, 20) = 7.217, p = 0.0142; adult life adversity effect: F(1, 20) = 55.44, p < 0.0001; early × adult life adversity: F(1, 20) = 0.4967, p = 0.4891; inset of Fig. 3b). Using two-way ANOVA followed by Tukey’s post hoc test implied no significant difference in the plasma leptin levels among the animals (early life adversity effect: F(1, 20) = 1.350, p = 0.259; adult life adversity effect: F(1, 20) = 1.159, p = 0.294; early × adult life adversity: F(1, 20) = 5.737, p = 0.0265; Fig. 3c).

Fig. 3.

Plasma CORT levels at weaning (PND 21) (a) and during young adulthood (b) and plasma leptin levels (c). Bars represent the mean ± SEM (6 rats/group). *p< 0.05 or less versus Std-Con group, p< 0.05 or less versus MS-Con group. Std-Con, rats under normal conditions; MS-Con, maternal separated rats; Std-CSDS, rats undergoing chronic social defeat stress; MS-CSDS, rats undergoing both maternal separation and chronic social defeat stress.

Fig. 3.

Plasma CORT levels at weaning (PND 21) (a) and during young adulthood (b) and plasma leptin levels (c). Bars represent the mean ± SEM (6 rats/group). *p< 0.05 or less versus Std-Con group, p< 0.05 or less versus MS-Con group. Std-Con, rats under normal conditions; MS-Con, maternal separated rats; Std-CSDS, rats undergoing chronic social defeat stress; MS-CSDS, rats undergoing both maternal separation and chronic social defeat stress.

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Behavioral Analysis

Open-Field Test

Statistical analysis indicated that locomotor activity was significantly decreased in Std-CSDS and MS-CSDS animals compared to Std-Cons (p < 0.001) (early life adversity effect: F(1, 36) = 3.756, p = 0.0605; adult life adversity effect: F(1, 36) = 141.1, p < 0.0001; early × adult life adversity: F(1, 36) = 0.2342, p = 0.6313; Fig. 4a). Statistical analysis also illustrated a significant decrease among MS-CSDS compared to MS-Con animals (p < 0.001).

Fig. 4.

Behavioral tests. a Locomotor activity during a 10 min exposure to the OFT. b Percentage of sucrose consumed over the total amount of liquid consumed. c % social avoidance index. Bars represent the mean ± SEM (10 rats/group). *p< 0.05 or less versus Std-Con group, p< 0.05 or less versus MS-Con groups, +p< 0.05 or less versus Std-CSDS groups. Std-Con, rats under normal conditions; MS-Con, maternal separated rats; Std-CSDS, rats undergoing chronic social defeat stress; MS-CSDS, rats undergoing both maternal separation and chronic social defeat stress.

Fig. 4.

Behavioral tests. a Locomotor activity during a 10 min exposure to the OFT. b Percentage of sucrose consumed over the total amount of liquid consumed. c % social avoidance index. Bars represent the mean ± SEM (10 rats/group). *p< 0.05 or less versus Std-Con group, p< 0.05 or less versus MS-Con groups, +p< 0.05 or less versus Std-CSDS groups. Std-Con, rats under normal conditions; MS-Con, maternal separated rats; Std-CSDS, rats undergoing chronic social defeat stress; MS-CSDS, rats undergoing both maternal separation and chronic social defeat stress.

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Sucrose Preference Test

According to two-way analysis of variance, followed by Tukey’s post hoc test (early life adversity effect: F(1, 36) = 9.742, p = 0.0035; adult life adversity effect: F(1, 36) = 13.88, p = 0.0007; early × adult life adversity: F(1, 36) = 2.631, p = 0.1135; Fig. 4b), there was a significant reduction in the percentage of sucrose consumed over the total amount of liquid consumed, which was considered as a sign of anhedonic behavior among the Std-CSDS rats compared to the Std-Con (p < 0.001) and MS-CSDS (p < 0.01) rats whereas no significant differences were observed between MS-Con, MS-CSDS, and Std-Con animals.

Social Avoidance Test

Using two-way ANOVA followed by Tukey’s post hoc test indicated a significant increase in %social avoidance index among the Std-CSDS rats compared to Std-Con rats (p < 0.05) whereas no significant differences were observed between MS-Con, MS-CSDS, and Std-Con animals (early life adversity effect: F(1, 36) = 2.045, p = 0.1613; adult life adversity effect: F(1, 36) = 9.949, p = 0.0032; early × adult life adversity: F(1, 36) = 2.416, p = 0.1288; Fig. 4c).

Body Weight, Food Intake, Lee Index, and BMI

Two-factor mixed-model ANOVA followed by the Tukey’s test for body weight (early life adversity effect: F(1, 12) = 3.906, p = 0.0716; time main effect: F(19, 228) = 871.4, p < 0.0001; early life adversity × time: F(19, 228) = 2.807, p = 0.0001; Fig. 5a), food intake (early life adversity effect: F(1, 12) = 0.3218, p = 0.0716; time main effect: F(4, 48) = 196.5, p < 0.0001; early life adversity effect × time: F(4, 48) = 2.470, p = 0.0571; Fig. 5b), Lee index (early life adversity effect: F(1, 12) = 0.3017, p = 0.5929; time main effect: F(7, 84) = 21.06, p < 0.0001; early life adversity effect × time: F(7, 84) = 0.9012, p = 0.5096; Fig. 5c), and BMI (early life adversity effect: F(1, 12) = 9.528, p = 0.0094; time main effect: F(7, 84) = 172.2, p < 0.0001; early life adversity × time: F(7, 84) = 0.7013, p = 0.6708; Fig. 5d) implied no significant differences between Std and MS offspring until PND 49. However, the calculated AUC for body weight (t12 = 2.559, p = 0.0251) and BMI (t12 = 3.188, p = 0.0078) revealed a significant decrease among MS offspring compared to Stds.

Fig. 5.

Body weight (a), food intake (b), Lee index (c), and BMI (d). Insets indicate the area under the curves. Bars represent the mean ± SEM (7 rats/group). *p< 0.05 or less versus Std-Con group, p< 0.05 or less versus MS-Con group. Std-Con, rats under normal conditions; MS-Con, maternal separated rats; Std-CSDS, rats undergoing chronic social defeat stress; MS-CSDS, rats undergoing both maternal separation and chronic social defeat stress.

Fig. 5.

Body weight (a), food intake (b), Lee index (c), and BMI (d). Insets indicate the area under the curves. Bars represent the mean ± SEM (7 rats/group). *p< 0.05 or less versus Std-Con group, p< 0.05 or less versus MS-Con group. Std-Con, rats under normal conditions; MS-Con, maternal separated rats; Std-CSDS, rats undergoing chronic social defeat stress; MS-CSDS, rats undergoing both maternal separation and chronic social defeat stress.

Close modal

Three-factor mixed-model ANOVA followed by the Tukey’s test (time effect: F(2, 48) = 34.623, p < 0.0001; early life adversity × time: F(1, 18) = 5.502, p = 0.007; adult life adversity × time: F(1, 18) = 5.323, p = 0.008; early × adult life adversity × time: F(3, 54) = 0.097, p = 0.908; early life adversity effect: F(1, 6) = 98.385, p < 0.0001; adult life adversity effect: F(1, 6) = 11.068, p = 0.003; early × adult life adversity: F(3, 18) = 4.931, p = 0.036; Fig. 5a) illustrated that Std-CSDS and MS-CSDS animals gained significantly less weight than Std-Cons on PND 56 onward. Using the same statistical tools mentioned above, the result of food intake (time effect: F(2, 48) = 4.462, p = 0.017; early life adversity × time: F(1, 18) = 9.905, p < 0.0001; adult life adversity × time: F(1, 18) = 1.913, p = 0.159; early × adult life adversity × time: F(3, 54) = 7.455, p = 0.002; early life adversity effect: F(1, 6) = 126.014, p < 0.0001; adult life adversity effect: F(1, 6) = 3.008, p = 0.096; early × adult life adversity: F(3, 18) = 0.103, p = 0.751; Fig. 5b), Lee index (time effect: F(2, 48) = 6.657, p = 0.003; early life adversity × time: F(1, 18) = 4.719, p = 0.013; adult life adversity × time: F(1, 18) = 1.691, p = 0.195; early × adult life adversity × time: F(3, 54) = 2.857, p = 0.067; early life adversity effect: F(1, 6) = 39.153, p < 0.0001; adult life adversity effect: F(1, 6) = 4.371, p = 0.047; early × adult life adversity: F(3, 18) = 9.860, p = 0.004; Fig. 5c), and BMI (time effect: F(2, 48) = 0.302, p = 0.741; early life adversity × time: F(1, 18) = 4.269, p = 0.020; adult life adversity × time: F(1, 18) = 2.800, p = 0.071; early × adult life adversity × time: F(3, 54) = 1.763, p = 0.182; early life adversity effect: F(1, 6) = 70.149, p < 0.0001; adult life adversity effect: F(1, 6) = 8.628, p = 0.007; early × adult life adversity: F(3, 18) = 10.084, p = 0.004; Fig. 5d) demonstrated that adult life adversity significantly suppressed food intake, lee index, and BMI in Std-CSDS and MS-CSDS animals in comparison with those under normal conditions, respectively, on PND 56 and PND 62 onward. The calculated AUC confirmed the mentioned reduction (insets of Fig. 5).

Percentage of Adrenal, Thymus, and Intra-Abdominal Fat Weights over the Body Weight

Percentage of adrenal weight/body weight significantly increased in MS-CSDS rats compared to Std-Con (p < 0.001) and MS-Con (p < 0.05) rats (early life adversity effect: F(1, 24) = 8.308, p = 0.0082; adult life adversity effect: F(1, 24) = 14.77, p = 0.0008; early × adult life adversity: F(1, 24) = 92.31, p = 0.3462; Table 2). Percentage of thymus weight/body weight was significantly increased among MS-CSDS (p < 0.01) and Std-CSDS (p < 0.05) rats compared to Std-Con rats (early life adversity effect: F(1, 24) = 1.405, p = 0.2476; adult life adversity effect: F(1, 24) = 15.61, p = 0.0006; early × adult life adversity: F(1, 24) = 84.97, p = 0.3658; Table 2). There was a significant decrease in the % intra-abdominal fat weight/body weight in Std-CSDS rats compared to Std-Cons (p < 0.01) (early life adversity effect: F(1, 24) = 0.0008, p = 0.9766; adult life adversity effect: F(1, 24) = 13.72, p = 0.0011; early × adult life adversity: F(1, 24) = 4.179, p = 0.0520; Table 2).

Table 2.

Percentage of adrenal, thymus, and intra-abdominal fat weights over the body weight (7 rats/group)

 Percentage of adrenal, thymus, and intra-abdominal fat weights over the body weight (7 rats/group)
 Percentage of adrenal, thymus, and intra-abdominal fat weights over the body weight (7 rats/group)

Oral Glucose Tolerance Test

Exposure to either early or adult life adversity did not significantly affect plasma glucose levels among animals (time effect: F(3, 60) = 22.234, p < 0.0001; early life adversity × time: F(1, 20) = 0.423, p = 0.737; adult life adversity × time: F(1, 20) = 1.965, p = 0.129; early × adult life adversity × time: F(3, 60) = 0.120, p = 0.948; early life adversity effect: F(1, 5) = 4.344, p = 0.050; adult life adversity effect: F(1, 5) = 0.445, p = 0.512; early × adult life adversity: F(3, 15) = 0.123, p = 0.729; Fig. 6a). However, as shown in Figure 6b, a significant increase of insulin response was observed among MS-CSDS (p < 0.01) animals 120 min after glucose injection (time effect: F(3, 60) = 12.996, p < 0.0001; early life adversity × time: F(1, 20) = 0.526, p = 0.666; adult life adversity × time: F(1, 20) = 1.190, p = 0.321; early × adult life adversity × time: F(3, 60) = 0.246, p = 0.864; early life adversity effect: F(1, 5) = 8.470, p = 0.009; adult life adversity effect: F(1, 5) = 4.818, p = 0.040; early × adult life adversity: F(3, 15) = 0.032, p = 0.860). The calculated AUC confirmed the increase of insulin response in MS-CSDS rats compared to Std-Cons (early life adversity effect: F(1, 20) = 3.513, p = 0.0756; adult life adversity effect: F(1, 20) = 7.199, p = 0.0143; early × adult life adversity: F(1, 20) = 0.0046, p = 0.9464; inset of Fig. 6b).

Fig. 6.

Plasma glucose (a) and insulin (b) levels in fasting state (0 min) and during oral glucose tolerance test (OGTT). Insets indicate the area under the curves. Bars represent the mean ± SEM (6 rats/group). *p< 0.05 or less versus Std-Con group. Std-Con, rats under normal conditions; MS-Con, maternal separated rats; Std-CSDS, rats undergoing chronic social defeat stress; MS-CSDS, rats undergoing both maternal separation and chronic social defeat stress.

Fig. 6.

Plasma glucose (a) and insulin (b) levels in fasting state (0 min) and during oral glucose tolerance test (OGTT). Insets indicate the area under the curves. Bars represent the mean ± SEM (6 rats/group). *p< 0.05 or less versus Std-Con group. Std-Con, rats under normal conditions; MS-Con, maternal separated rats; Std-CSDS, rats undergoing chronic social defeat stress; MS-CSDS, rats undergoing both maternal separation and chronic social defeat stress.

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HOMA-IR, QUICKI, and ISI Indices and %HOMA-B

Statistical analysis implied no marked difference in HOMA-IR index (early life adversity effect: F(1, 20) = 1.284, p = 0.2705; adult life adversity effect: F(1, 20) = 12.56, p = 0.7267; early × adult life adversity: F(1, 20) = 0.0002, p = 0.9879; Table 3), QUICKI (early life adversity effect: F(1, 20) = 0.1220, p = 0.7306; adult life adversity effect: F(1, 20) = 0.1220, p = 0.7306; early × adult life adversity: F(1, 20) = 0.1220, p = 0.7306; Table 3), and ISI (early life adversity effect: F(1, 20) = 4.545, p = 0.0456; adult life adversity effect: F(1, 20) = 1.537, p = 0.2294; early × adult life adversity: F(1, 20) = 0.0329, p = 0.8578; Table 3) between the study groups. However, %HOMA-B (early life adversity effect: F(1, 20) = 4.144, p = 0.0552; adult life adversity effect: F(1, 20) = 4.990, p = 0.0371; early × adult life adversity: F(1, 20) = 0.8547, p = 0.3662; Table 3) was significantly increased in MS-CSDS animals compared to Std-Cons (p < 0.05).

Table 3.

HOMA-IR, QUICKI, ISI, and %HOMA-β (6 rats/group)

 HOMA-IR, QUICKI, ISI, and %HOMA-β (6 rats/group)
 HOMA-IR, QUICKI, ISI, and %HOMA-β (6 rats/group)

Glucose-Stimulated Insulin Secretion and Content of Pancreatic Isolated Islets

Three-way ANOVA followed by the Tukey’s test (early life adversity effect: F(1, 3) = 64.235, p < 0.0001; adult life adversity effect: F(1, 3) = 31.363, p < 0.0001; glucose concentration effect: F(1, 12) = 511.432, p < 0.0001; early × adult life adversity: F(3, 9) = 63.458, p < 0.0001; early life adversity × glucose concentration: F(1, 6) = 23.632, p < 0.0001; adult life adversity × glucose concentration: F(1, 6) = 10.399, p = 0.004; early × adult life adversity × glucose concentration: F(3, 18) = 0.203, p = 0.656; Table 4) illustrated that GSIS of isolated islets (pmol/mL) was significantly decreased in MS-CSDS animals compared to Std-Con rats in the presence of 16.7 mM of glucose concentration (p < 0.001). There is also a significant decrease in GSIS of isolated islets in the presence of both glucose concentrations among MS-CSDS rats than MS-CSDS and Std-CSDS groups (p < 0.001). Using the same statistical tools for the insulin content of isolated islets (µg/mg protein) (early life adversity effect: F(1, 3) = 14.854, p = 0.001; adult life adversity effect: F(1, 3) = 0.273, p = 0.606; glucose concentration effect: F(1, 12) = 154.827, p < 0.0001; early × adult life adversity: F(3, 9) = 0.017, p = 0.897; early life adversity × glucose concentration: F(1, 6) = 6.805, p = 0.015; adult life adversity × glucose concentration: F(1, 6) = 0.578, p = 0.454; early × adult life adversity × glucose concentration: F(3, 18) = 0.207, p = 0.653; Table 4) indicated a significant decrease in the presence of 16.7 mM of glucose concentration among Std-CSDS (p < 0.05) and MS-CSDS (p < 0.01) rats than Std-Cons.

Table 4.

Insulin secretion (pmol/mL) and insulin content (µg/mg protein) of pancreatic isolated islets in response to 5.6 and 16.7 mM of glucose concentrations (4 rats/group, two groups of ten islets for each concentration of glucose in each animal)

 Insulin secretion (pmol/mL) and insulin content (µg/mg protein) of pancreatic isolated islets in response to 5.6 and 16.7 mM of glucose concentrations (4 rats/group, two groups of ten islets for each concentration of glucose in each animal)
 Insulin secretion (pmol/mL) and insulin content (µg/mg protein) of pancreatic isolated islets in response to 5.6 and 16.7 mM of glucose concentrations (4 rats/group, two groups of ten islets for each concentration of glucose in each animal)

Histological Analysis of the Pancreatic Islet Tissues

Two-way analysis of variance followed by Tukey’s post hoc test (early life adversity effect: F(1, 315) = 113.1, p < 0.0001; adult life adversity effect: F(1, 315) = 1231, p < 0.0001; early × adult life adversity: F(1, 315) = 120.4, p < 0.0001; Fig.7b) illustrated a significant decrease in the β-cell number as a percentage of the area of islets in animals who were exposed to either adversity during early life (MS-Con), adulthood (Std-CSDS), or both of them (MS-CSDS) compared to Std-Cons (p < 0.001). There is also a significant difference between the MS-CSDS and MS-Con groups (p < 0.001).

Fig. 7.

Histological findings of pancreas tissue after the combination of aldehyde fuchsin and H&E staining in different experimental groups (a) and %β-cell number/area of islets (b). a, Std-Con: rats under normal conditions; b, MS-Con: maternal separated rats; c, Std-CSDS: rats undergoing chronic social defeat stress; d, MS-CSDS: rats undergoing both maternal separation and chronic social defeat stress. Arrows show β cells in the islets of Langerhans containing insulin granules that are strongly stained in deep purple-violet (×40). Bars represent the mean ± SEM (2 rats/group). *p< 0.05 or less versus Std-Con group, p< 0.05 or less versus MS-Con group.

Fig. 7.

Histological findings of pancreas tissue after the combination of aldehyde fuchsin and H&E staining in different experimental groups (a) and %β-cell number/area of islets (b). a, Std-Con: rats under normal conditions; b, MS-Con: maternal separated rats; c, Std-CSDS: rats undergoing chronic social defeat stress; d, MS-CSDS: rats undergoing both maternal separation and chronic social defeat stress. Arrows show β cells in the islets of Langerhans containing insulin granules that are strongly stained in deep purple-violet (×40). Bars represent the mean ± SEM (2 rats/group). *p< 0.05 or less versus Std-Con group, p< 0.05 or less versus MS-Con group.

Close modal

Early and/or adult life adversity decreased %β-cell number/area of islets, muscular FKBP51 content, and BMI in young adulthood. The reduction of %β-cell number/area of islets and BMI in the MS-CSDS rats were more profound than MS-Con group. Exposure to CSDS either alone or in combination with MS reduced locomotor activity and increased and decreased CRFR1 content in hypothalamus and pancreas. Moreover, under CSDS, MS exacerbated HPA axis overactivity (manifested by increased CORT secretion, %adrenal weight/body weight, and hypothalamic FKBP51 and CRFR1 protein contents) and reduced GSIS of isolated islets. Meanwhile, it could promote resilience to symptoms of depression. However, no changes in regional DNA methylation status of the hypothalamic Fkbp5 gene promoter and glucose tolerance were observed among groups.

In agreement with previous studies, our findings showed that Std-CSDS animals, on average, displayed increased basal CORT circulating levels, anhedonia, social avoidance, and decreased locomotor activity, reminiscent of susceptibility to develop depressive-like behavior [6, 39, 51]. Furthermore, the elevated hypothalamic CRFR1 and FKBP51 contents in response to chronic social stress in Std-CSDS and MS-CSDS animals was in accordance with previous findings in which chronic exposure to CORT or mild stressor has been stated to increase Fkbp5 or Crfr1 expression in several structures of brain including hypothalamus [52‒55]. We should note, though, the increase in CORT levels was along with increases of %adrenal weight/body weight and the hypothalamic protein contents of FKBP51 and CRFR1 of bulk hypothalamic tissue, reminiscent of HPA axis overactivity, aiming PVN, as a more specific region in HPA axis regulation, would be important to be considered in future studies. In this study, no changes in regional DNA methylation status of the hypothalamic Fkbp5 gene promoter were observed under chronic exposure to social defeat. Contrary to our finding, Lee et al. [53] found that the elevated level of Fkbp5 expression under chronic exposure to CORT via drinking water (100 μg/mL for 4 weeks) was accompanied with decreased DNA methylation of the gene in hypothalamus of C57BL/6J mice. This discrepancy might be related to the differences of the animal species or different experimental paradigms employed to increase plasma CORT levels. Moreover, we assessed DNA methylation status in only one small representative region of Fkbp5 gene promoter; however, the use of a comprehensive genome-wide assay could perhaps reveal the methylation changes of the gene. It is also worth noting that central Fkbp5 and Crfr1 alterations have been suggested as keys to the gene-environment interactions that not only change normal HPA axis functionality but also contribute to the stress-related behavioral changes, such as depression and anxiety disorders [56‒59]. In this vein, two laboratories have constructed Fkbp5 knockout mice that showed more active stress-coping behavior in the forced swim test and lower CORT response to acute or chronic stress and improved resilience to the CSDS paradigm compared to wild-type litter mates [60, 61]. However, unlike the intensified HPA axis overactivity (represented by increased basal CORT secretion, percentage of adrenal weight over body weight, and hypothalamic FKBP51 and CRFR1 protein contents) in MS adult rats under CSDS, on average, they indicated resilience to anhedonia and social avoidance, as main symptoms of depression. It was in accordance with our recent work [62] in which we revealed that MS could promote resilience to the passive stress-coping style (immobility), independent of CORT elevation in response to CSDS. Reports on the effects of ELS on neuroendocrine responses to subsequent stressors and vulnerability to behavioral disorders are conflicting. Indeed, while a seemingly paradoxical protective effect of ELS to develop subsequent stress resistance “match/mismatch hypothesis” has been reported by some authors [38, 63], another reported that it increases the vulnerability to further challenges in later “cumulative hypothesis” [3]. Generally, in animals raised under ELS, behavioral resilience to adulthood stressful conditions has been reported to simultaneously occur with a dampened overall HPA axis activity [64‒66]. However, in accordance with our findings, Pérez-Tejada et al. [67] reported that mice with active and passive phenotypes based on the immobility behavior and social exploration had similar increases in CORT and hypothalamic corticotropin-releasing factor levels in response to chronic social stress. Similarly, Krishnan et al. (2007) [68] reported that mice who were resilient to anhedonia displayed an elevated CORT reactivity and anxiety at the end of the CSDS. Accordingly, it would make sense that MS could shape the growing individual’s behavior to cope with later social challenges, independent of HPA axis hyperactivity. In addition, we have also suggested that compensatory elevated levels of maternal care, following reuniting the separated dams and pups, probably contributed to the development of resilience to depressive-like behavior under later stress in adult MS offspring [62].

It is noteworthy that a synchronicity between depression and metabolic disorders, such as diabetes, has been recognized over the years [22, 69]. This association was another motivation encouraging us to investigate the effects of MS on energy and glucose homeostasis in response to later social challenges. The present study provided new evidence that the protein content of CRFR1 in the pancreas, along with %β cell-number/area of islets and the insulin content of isolated islets, were markedly decreased under chronic exposure to social defeat stress, in Std-CSDS and MS-CSDS rats. It has been revealed that CRFR1 promotes β-cell survival [19] and function [21] in pancreatic islets. There is also ample evidence that GR expresses in β cells [70] and their hyperactivity directly impairs β-cell viability and function [71]. Meanwhile, several in vitro studies illustrated the marked inhibitory effect of cortisol, prednisolone, or dexamethasone on GSIS of pancreatic islets [72, 73]. In this sense, our results raise the possibility that a significant reduction of pancreatic CRFR1 content besides elevated CORT levels contributed to progressive β-cell loss under chronic social challenges in adulthood. Furthermore, we recently revealed a significant increase in pancreatic oxidative and inflammatory damage markers (malondialdehyde and interleukin-1β) in MS-CSDS rats [74], which along with an excessive basal level of circulating CORT and diminished pancreatic CRFR1 content are likely to involve in the reduction of β-cell function and insulin secretion capacity. Contrary to our findings, Sadeghimahalli et al. (2015) [75] demonstrated that electrical shock stress at both postnatal and young adulthood increased GSIS from pancreatic isolated islets in rats. Additionally, it has been reported that MS alone (from PND 1–21, 3 h daily) could reduce the insulin content without affecting the insulin secretion capacity of pancreatic isolated islets [76]. The different results could be attributed to the different stress paradigm being used, including type, onset, length, and duration of stress. Growing evidence has emphasized the role of high concentrations of glucocorticoids in the induction of insulin resistance directly by decreasing the recruitment of GLUT4 to the cell membranes, reducing insulin receptor substrate-1 expression, and damaging phosphatidylinositol-3 kinase and protein kinase C or indirectly by inducing pro-inflammatory factors [77]. Additionally, it has been previously indicated that higher levels of FKBP51, through inactivation of AKT2-AS160 signaling and inhibition of GLUT4 translocation, ultimately lower glucose uptake in primary myotubes, which suggesting a role for FKBP51 in the insulin resistance development under stressful conditions [18]. However, in contrast to our hypothesis, the current study provided new evidence that early and/or adult life adversity reduced muscular FKBP51 content and it is likely to be a compensatory response to slow down insulin resistance development under CORT hypersecretion in stressful challenges. Nevertheless, this hypothesis needs further investigations. Unlike the unchanged indices of insulin resistance and plasma glucose levels, insulin response 120 min after glucose injection increased significantly in MS-CSDS animals. The significantly higher insulin AUC confirms the increased insulin response to glucose injection in MS-CSDS animals. Considering the significant increase in insulin response to glucose loading during OGTT to maintain normal plasma glucose levels, the onset of insulin resistance could not be ruled out in MS-CSDS rats. Several lines of evidence have indicated that adequate plasma insulin is determined through a cross-talk between pancreatic β-cell secretion and hepatic insulin clearance to maintain normal glucose tolerance [78, 79]. Since the isolated islet’s insulin secretion and content in response to glucose were significantly decreased in MS-CSDS rats, the increase in calculated %HOMA-β, as an index of β-cell function, could not explain the observed elevated insulin response during OGTT. Consequently, the increase in plasma insulin levels during OGTT might be due to alterations in hepatic insulin clearance. In this association, it has been revealed that carcinoembryonic antigen-related cell adhesion molecule (CEACAM1) involved in the receptor-mediated uptake of insulin in the hepatocytes [80] and insulin degrading enzyme, which degrades insulin in the endosome, affect insulin clearance [81]. Of note, insulin degrading enzyme was reported to be upregulated by insulin [82] while CEACAM1 expression was diminished using HF diet as a metabolic stressor in C57/BL6J mice [83]. Meanwhile, prenatal stress (restraint stress, three times/day from days 14 to 21 of gestation) was reported to decrease hepatocyte CEACAM1 levels in diet-induced obese offspring significantly after the high fat challenge [84]. In this regard, the increased insulin response to glucose injection in MS-CSDS rats is likely to be a consequence of reduced first-pass insulin extraction as a compensatory response to insulin secretion reduction and/or programming alterations of the abovementioned involved molecules that needs to be further elucidated in the future. As a next step, it will be intriguing to investigate the alterations of GR protein expression in peripheral metabolically relevant tissues (e.g., pancreas and skeletal muscle) as well as PVN of hypothalamus besides GR (NR3C1) gene DNA methylation changes in relation to impairment of insulin secretion capacity and incidence of insulin resistance under chronic stressful conditions.

In the current study, chronic exposure to social defeat stress significantly attenuated body weight gain, Lee index, BMI, intra-abdominal fat weight, and food intake, which was in accordance with previous experiments [85]. Body weight gain was also decreased in response to early life adversity that might be related to various factors, namely, the nutritional deficit, maternal milk secretion due to reduced suckling stimulation of pups, or plasma growth hormone suppression during separation [86]. To elucidate the reason for suppressed food intake, we evaluated the levels of peripheral leptin. Contrary to the diminished plasma leptin levels reported in previous animal models of stress [87‒89], we did not find any significant changes in peripheral leptin levels. Normally, leptin and blood glucose increase the level of malonyl-CoA in the hypothalamus, suppressing food intake by changing hypothalamic expression of numerous neuropeptides associated with feeding behavior [90]. It has been proposed that chronic stress may perturb the regulation of hypothalamic malonyl-CoA and in turn food intake, independent of leptin levels [91]. Additionally, given the strong corticotropin-releasing factor anorectic effects, the hyperactivation of these neurons in PVN under chronic exposure to stress has been suggested to contribute in the stress-induced appetite reduction in the male chow-fed rats [92].

Taking into account the results obtained in the present work, it seems that in the presence of MS, the incidence of HPA axis overactivity and the endocrine pancreas impairments in response to later exposure to stress was intensified. The reduced pancreatic CRFR1 protein content and the intensified elevated plasma CORT levels might be the reasons for the reduced β-cell number and insulin secretion capacity in MS adult rats under CSDS. Herein, we also implied that the decreased muscular FKBP51 content might be a homeostatic response to slow down development of insulin resistance under CORT hypersecretion in stressful conditions.

This article has been extracted from the thesis written by Mrs. Farzaneh Eskandari in School of Medicine, Shahid Beheshti University of Medical Sciences (Registration No.: 57).

The procedures involving animal subjects were carried out in compliance with the National Institutes of Health Guide for the care and use of laboratory animals and were reviewed and approved by the Ethics Committee of Shahid Beheshti University of Medical Sciences (ethic code: IR.SBMU.MSP.REC.1397.440).

The authors have no conflicts of interest to declare.

This work has been supported financially by the Neurophysiology Research Center, Shahid Beheshti University of Medical Sciences (Grant No. 16909).

Farzaneh Eskandari: investigation, methodology, formal analysis, writing – original draft, writing – review and editing, and visualization. Mina Salimi: investigation and methodology. Fateme Binayi: investigation. Mohammad-Amin Abdollahifar: methodology and resources. Mohamad Eftekhary: methodology and validation. Mehdi Hedayati: validation, data curation, and resources. Hossein Ghanbarian: methodology, data curation, and resources. Homeira Zardooz: conceptualization, visualization, supervision, data curation, project administration, writing – review and editing, and funding acquisition.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

1.
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