Investigation of the effects of maternal separation on the pancreatic oxidative and inflammatory damages along with metabolic impairment in response to chronic social defeat stress in young adult male rats

Abstract

Purpose

Chronic glucocorticoid release during the stress response has been proposed to initiate certain damages, which in turn produce metabolic disorders. The present study is the first work to test whether maternal separation (MS) would impact the metabolic alterations associated with pancreatic oxidative and inflammatory damages under chronic exposure to social defeat stress (CSDS) in adulthood.

Methods

During the first 2 weeks of life, male Wistar rats were 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. Thus, there were 4 groups (n = 7/group): Std-Con, Ms-Con, Std-CSDS, MS-CSDS. Each animal was weighed and then decapitated so that we could collect trunk blood for assessment of fasting plasma corticosterone, insulin, glucose, lipid profile, and insulin resistance. Plasma and pancreatic catalase activity, reduced glutathione (GSH), malondialdehyde levels and pancreatic interleukin-1 beta (IL-1β) content were also measured.

Results

MS-CSDS animals showed elevated plasma corticosterone and insulin levels (P < 0.01) along with insulin resistance (P < 0.05). According to one-way ANOVA results, chronic exposure to early or adult life adversity decreased body weight (P < 0.0001), Catalase activity and GSH levels (P < 0.0001) and increased malondialdehyde level (P = 0.0006) in plasma. Pancreatic MDA and IL-1β contents elevated just in MS-CSDS rats (P < 0.05).

Conclusion

Maternal separation shapes vulnerability to develop corticosterone hypersecretion, insulin resistance, pancreatic oxidative, and inflammatory damages associated with chronic exposure to later social challenges, which could potentially trigger metabolic disorders.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40200-021-00902-3.

Introduction

Accumulating evidence revealed that a range of factors, such as early experiences and social situations, shape individual differences in stress reactivity and vulnerability to disease in future life [1]. Negative experiences in early life, such as parental loss, abuse, and being emotionally or physically neglected, are effective factors that may shape individual differences in stress reactivity and increase the risk of developing chronic degenerative diseases later in life [2]. In rodents, during the first two weeks of life, pups have been reported to indicate a weakened HPA axis reactivity to mild stressors (stress hypo-responsive period; SHRP) to protect the developing brain from fast and excessive changes in circulating corticosterone level [3]. Therefore, separating pups from the dam by disturbing SHRP exerts persistent alterations of structural and neuroendocrine responses to stress in adult offspring [4]. Moreover, since humans are mostly exposed to psychological or social stressors at adult ages [5], social defeat stress, as an animal model of social conflicts among members of the same strains, could be an ethologically valid stressor to be used in adult animals [6]. Under chronic exposure to stress, homeostasis reestablishment or allostasis load involves HPA axis hyperactivity, considered as a major risk factor for metabolic alterations [7, 8]. In this sense, corticosterone hypersecretion induces oxidative stress and metabolic dysregulation (hyperglycemia or insulin resistance) [9, 10], which in turn contribute to later pancreatic β-cells impairments and the risk of diabetes development [11, 12]. Furthermore, it has been revealed that the pancreatic β-cells possess a relatively weak antioxidant system and the disruption of ROS homeostasis can easily induce oxidative stress in these cells [13, 14]. For note, chronic psychological stress has been reported to induce high levels of malondialdehyde (MDA), as oxidative damage marker, in patients with newly diagnosed diabetes mellitus [15]. Oxidative stress also elevates the proinflammatory cytokines levels [16], among which interleukin-1 beta (IL-1β) has been indicated to increase the number of apoptotic pancreatic β-cells [17] and incidence of diabetes [18].

According to the points mentioned above, we aimed to explore whether early life adversity represented by maternal separation (MS) impacts the possible negative effects of the adulthood chronic social defeat stress (CSDS), on the onset of metabolic impairments associated with oxidative stress and inflammation. To this end, among essential nonenzymatic and enzymatic cellular antioxidants, we assessed glutathione (GSH, l-γ-glutamyl-l-cysteinylglycine) content and catalase (CAT) activity, in plasma and pancreas. Moreover, we investigated the probable alterations in systemic and pancreatic MDA contents as well as pancreatic IL-1β.

Materials and methods

Animals

Procedures involving animal subjects were approved by the Institutional Animal Care and Ethics Committee of Shahid Beheshti University of Medical Sciences (Ethics Code No. IR.SBMU.REC.1397.103) and were executed in compliance with the national institutes of Health Guide for the care and use of laboratory animals.

Female pregnant Wistar rats (200 ± 20 g) were individually kept under standard laboratory conditions (12:12 light/dark cycle, 22 °C, food and water ad libitum) until delivery. The day of birth was termed as postnatal day 0 (PND 0). On PND 1, the female pups were removed and the males were remained with the dam. It is noteworthy that the litters were culled to 3–5 male pups (For more details see supplementary materials).

Experimental design

In this study, there were four groups (7 rats/group, 5 litters/group): standard-reared control rats (Std-Con), maternal separated rats (MS-Con), rats underwent chronic social defeat stress (Std-CSDS) and those exposed to both maternal separation and chronic social defeat stress (MS-CSDS) (Fig. 1).

Fig. 1.

Experimental procedures over the course of the experiment. Std standard-rearing, Con no adult stress, MS maternal separation, CSDS chronic social defeat stress. (7 rats/group, 5 litters/group)

Male pups were exposed to 3-h of daily maternal separation (MS) at random times or left undisturbed with their mothers (Std) during the first 2 weeks of life (14 pups/group, 5 litters/group). After PND 14, pups and their mothers were left undisturbed until they were weaned on PND 21. Offspring were then randomly grouped (into two to three per cage) according to neonatal exposure to stress (MS) or standard-rearing (Std) and left undisturbed until puberty (PND50). Then, the animals of each group were either left undisturbed in standard housing conditions (Con) or underwent chronic social defeat stress (CSDS) for 3 weeks. The animals of Std-CSDS and MS-CSDS subgroups daily replaced with cohabitating females in the home cage of the aggressive male residents [19]. The intruder experienced 10 min physical contact followed by 1-h psychogenic exposure to the resident (For more details see supplementary materials).

Blood sampling and tissue collection

On PND 72, the overnight fasted (16–18 h) animals were weighted (7 rats/group, 5 litters/group) and were briefly exposed to isoflurane (Nicholas Primal, London, UK) as an anaesthetic agent. Each animal was decapitated and trunk blood was collected into tubes containing heparin (5000 IU/ml) (10 μl/ml) and centrifuged at 664×g for 10 min to obtain plasma. Plasma aliquots were stored at –80 °C until biochemical analyses of plasma parameters and oxidative assays. Then the pancreas was quickly removed and transferred to liquid nitrogen and stored at − 80 °C. The pancreas samples were homogenized in the lysis buffer and centrifuged at 12,000×g for 30 min at 4 °C. The supernatant was collected and stored at − 80 °C until analysis of total protein concentration and other assays. Moreover, the intra-abdominal fat was removed and weighed.

Biochemical kits

Plasma corticosterone concentration was determined using a rat corticosterone ELISA kit (sensitivity: 0.9 nmol/l) (ZellBio, Germany). The intra-assay coefficient of variations was 6.1%.

Plasma glucose concentration was 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 (sensitivity: 0.1 mIU/L) (ZellBio, Germany). The intra-assay coefficient of variations was 5.1%.

Plasma triglyceride (TG) and total cholesterol (TC) levels, were determined using an Enzymatic Photometric kit (Parsazmun, Tehran, Iran). The sensitivity of the assay for the TG and TC were 1 mg/dl and 3 mg/dl, respectively.

Calculation of insulin resistance index

An updated homeostasis model assessment of insulin resistance (HOMA2-IR) as a surrogate marker of IR and a useful non-invasive way of determining insulin sensitivity were calculated using fasting glucose and fasting insulin by HOMA calculator for specific insulin, version 2.2.3 available from http://www.dtu.ox.ac.uk/ homacalculator [20].

Pancreatic supernatant protein assay

The total protein concentration of pancreatic supernatant was estimated based on the method of Bradford [21]. A standard curve was generated using bovine serum albumin as the standard and all biochemical measures were normalized to the protein content.

Plasma and pancreatic tissue oxidative assessments

CAT catalyzes the breakdown of H₂O₂ to H₂O and O₂. The rate of decomposition of H₂O₂ was measured spectrophotometrically using the method described by Goth [22] with some modification (For more details see Supplementary Materials).

The antioxidant GSH level was assayed according to the method of Ellman [23] (For more details see Supplementary Materials).

MDA as the most abundant toxic aldehyde formed from polyunsaturated fatty acids peroxidation, could be used as a measure for biological oxidative stress [24]. MDA levels were determined by using the commercial colorimetric kit (sensitivity: 0.1 µmol/l) (ZellBio, Germany) and calculated from the standard curve and expressed as nmol/mg protein (µmol/ml for plasma). The intra-assay coefficient of variations was 6.9%.

Pancreatic IL-1β assessment

IL-1β level of pancreas was measured using a rat ELISA kit (ZellBio, Germany) and the sensitivity of the assay was 3.5 pg/ml.

Statistical analyses

Analyses were done using the software Graph Pad Prism version 8.0 and all values are presented as mean ± standard error of mean. One-way analysis of variance (ANOVA) followed by Tukey post hoc test was used for analyzing data. Stress were set as independent factor. The level of confidence was set at 95% (P < 0.05 was considered statistically significant).

Results

The effect of maternal separation and/or CSDS on Body weight and intra-abdominal fat weight in young adult male rats

One-way analysis of variance followed by Tukey’s post hoc test [F(3,24) = 40.05, P < 0.0001; Fig. 2a], revealed that MS-Con, Std-CSDS and MS-CSDS animals had significantly lower body weight compared to Std-Cons; (P < 0.001) on PND 72. Intra-abdominal fat weight was also significantly lower among Std-CSDS (P < 0.001) and MS-CSDS (P < 0.01) rats compared to Std-Con rats. Figure 2b, also show a significant difference in the intra-abdominal fat weight between the MS-CSDS and MS-Con groups; (P < 0.01) [F(3,24) = 13.28, P < 0.0001].

Fig. 2.

Body weight (a) and intra-abdominal fat weight (b). Bars represent the mean ± SEM (7 rats/group, 5 litters/group). *P < 0.05, ***P < 0.001 versus Std-Con group, ^††P < 0.01 versus MS-Con group. Std-Con rats under normal condition, 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

Fasting corticosterone level

There was a significant increase in circulating corticosterone levels in the MS-CSDS group compared to the Std-Con (P < 0.01) and MS-Con (P < 0.001) groups. Nevertheless, no significant differences were observed between the animals of the MS-Con, Std-CSDS and Std-Con groups [F(3,20) = 9.102, P = 0.0005; Table 1].

Table 1.

Biochemical parameters of plasma and HOMA2-IR

Group	Corticosterone (nmol/l)	Glucose (mg/dl)	Insulin (pmol/l)	HOMA2-IR	Triglyceride (mg/dl)	Cholesterol (mg/dl)
Std-Con	519.96 ± 34.67	133.57 ± 9.02	192.85 ± 11.10	3.79 ± 0.17	50.65 ± 5.71	50.14 ± 1.20
MS-Con	511.55 ± 21.24	121.45 ± 6.57	214.39 ± 9.97	4.11 ± 0.20	55.93 ± 7.65	52.91 ± 0.71
Std-CSDS	593.21 ± 29.54	116.49 ± 1.57	212.43 ± 5.44	4.05 ± 0.10	43.34 ± 1.19	71.82 ± 3.69***
MS-CSDS	685.44 ± 18.34**,†††	108.27 ± 6.53	245.74 ± 13.10**	4.55 ± 0.23*	45.71 ± 3.25	64.82 ± 1.72**,†

All the data are expressed as the mean ± SEM (6 rats/group, 5 litters/group)

Std-Con: rats under normal condition, 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

*P < 0.05, **P < 0.01, ***P < 0.001 versus Std-Con group, ^†P < 0.05, ††† P < 0.001 versus MS-Con group

Fasting glucose level

According to one-way analysis of variance followed by Tukey's post hoc test, fasting plasma glucose level did not differ significantly between the groups [F(3,20) = 2.554, P = 0.0843; Table 1].

Fasting insulin level and index of insulin resistance (HOMA2-IR)

The combination of early and adult life adversity could significantly increase fasting plasma insulin level [F(3, 20) = 4.512, P = 0.0142; Table 1] and insulin resistance index [F(3, 20) = 3.097, P = 0.0501; Table 1] in the MS-CSDS animals (P < 0.01, plasma insulin; P < 0.05, HOMA2-IR) compared to Std-Con group.

Triglyceride and total cholesterol levels

According to one-way analysis of variance followed by Tukey's post hoc test, plasma triglyceride level did not differ significantly between the groups [F(3,20) = 2.030, P = 0.1421; Table 1]. While adulthood adverse experience significantly increased plasma total cholesterol level [F(3,20) = 15.18, P < 0.001; Table 1] among the Std-CSDS (P < 0.001) and MS-CSDS (P < 0.01) rats in comparison to Std-Con rats. There is also a significant difference in plasma total cholesterol level between the MS-CSDS and MS-Con groups; (P < 0.05).

Enzymatic and non-enzymatic antioxidants and lipid peroxidation marker in the pancreas and plasma

Catalase activity

The combination of MS and CSDS could significantly increase the activity of catalase in pancreatic tissue of MS-CSDS rats when compared with Std-Con rats (P < 0.01) and MS-Con rats (P < 0.05). However, early or adult life adversity alone had no distinguishable effect on the catalase activity in comparison with those raised under normal environments [F(3,16) = 7.634, P = 0.0022; Table 2].

Table 2.

Lipid peroxidation marker, enzymatic and non-enzymatic antioxidants in the pancreas (5 rats/group, 5 litters/group) and plasma (6 rats/group, 5 litters/group)

Group	Catalase activity		GSH level		MDA level
	Pancreas	Plasma	Pancreas	Plasma	Pancreas	Plasma
Std-Con	1.20 ± 0.09	577.67 ± 0.12	0.85 ± 0.09	13.92 ± 0.18	90.65 ± 7.64	4.18 ± 0.39
MS-Con	1.39 ± 0.07	315.97 ± 1.12*	1.28 ± 0.19	14.34 ± 0.15	115.75 ± 20.83	5.61 ± 0.25**
Std-CSDS	1.55 ± 0.21	20.80 ± 2.12***	1.74 ± 0.23*	7.56 ± 0.74***	97.80 ± 1.73	5.15 ± 0.15*
MS-CSDS	1.98 ± 0.02**,†	13.01 ± 3.12***†	3.34 ± 0.23***,†††,+++	10.07 ± 0.59***,†††,++	150.31 ± 16.55*	5.82 ± 0.23***

All the data are expressed as the mean ± SEM. CAT are expressed as µmole of H₂O₂ consumed/minute/mg protein for pancreatic tissue and µmole of H₂O₂ consumed/minute/l for plasma; GSH is expressed as µg/mg protein for pancreatic tissues and mg/dl for plasma; MDA is expressed as nmole/mg protein for pancreatic tissue and µmol/l for plasma. The assay was run in duplicate

Std-Con rats under normal condition, 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

*P < 0.05, **P < 0.01, ***P < 0.001 versus Std-Con group, ^†P < 0.05, †††P < 0.001 versus MS-Con group, ++P < 0.01, +++P < 0.001 versus Std-CSDS group

In contrast, the plasma catalase activity significantly reduced in MS-Con (P < 0.05), Std-CSDS (P < 0.001) and MS-CSDS (P < 0.001) rats in comparison with Std-Cons. There is also a significant difference in the plasma catalase activity between the MS-CSDS and MS-Con groups; (P < 0.05) [F(3,20) = 16.72, P < 0.0001; Table 2].[Edit]

GSH level

Chronic adversity during adult life caused a significant increase in the pancreatic GSH content of Std-CSDS (P < 0.05) and MS-CSDS (P < 0.001) rats in comparison with the Std-Con rats; [F(3, 16) = 30.42, P < 0.0001; Table 2]. In contrast, plasma GSH level significantly reduced in Std-CSDS and MS-CSDS rats when compared with Std-Con animals; (P < 0.001) [F(3, 20) = 44.25, P < 0.0001; Table 2].

Moreover, adult life adversity in combination with early adverse experience (MS-CSDS) significantly changed the GSH level in comparison to the adult (Std-CSDS) or early (MS-Con) life adversity alone; (P < 0.001).

MDA level

According to one-way analysis of variance followed by Tukey's post hoc test, pancreatic MDA content in MS-CSDS rats was significantly higher than in Std-Con rats; (P < 0.05). Nevertheless, no significant differences were observed between MS-Con, Std-CSDS and Std-Con animals [F(3,16) = 3.689, P = 0.03642; Table 2].

However, either early or adult life adversity could significantly increase the plasma MDA level in the MS-Con (P < 0.01), Std-CSDS (P < 0.05) and MS-CSDS (P < 0.001) rats in comparison with Std-Con rats [F(3,20) = 8.838, P = 0.0006; Table 2].

Pancreatic proinflammatory cytokine IL-1β content

There was a significant increase in IL-1β content of pancreatic tissue in the MS-CSDS rats compared to the Std-Con and Std-CSDS animals; (P < 0.05). Nevertheless, no significant differences were observed between MS-Con, Std-CSDS and Std-Con animals [F(3,16) = 4.312, P = 0.0208; Fig. 3].

Fig. 3.

IL-1β content in pancreas (5 rats/group, 5 litters/group). Bars represent the mean ± SEM. *P < 0.05 versus Std-Con, +P < 0.05 versus Std-CSDS groups. Std-Con rats under normal condition, 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

Discussion

The findings of this study revealed that the maternal separation shapes vulnerability to develop corticosterone hypersecretion, insulin resistance, pancreatic oxidative, and inflammatory damages in response to chronic social defeat stress in young adult male rats. Consistent with our findings, maternal separation has been reported to increase corticosterone response to postweaning social isolation stress [25] and adulthood chronic variable stress [26, 27]. The elevated corticosterone response to stress occurred in part due to the increased basal tone of central CRFergic activity and coincident intensified HPA axis responsiveness [28, 29]. It may be secondary to facilitation of noradrenergic neurocircuits [30], central glucocorticoid resistance [31], and/or dampened regional GABAergic tone [32], which have been observed to happen in response to maternal separation stress.

In accordance with well-documented evidence on the reduction in growth rate in rats, during and following psycho-social stress [33–35], we also found that body weights were significantly lower in animals under chronic social stress as well as early life stress compared to the standard-reared control (Std-Con) rats. Additionally, intra-abdominal fat weight was lower in Std-CSDS and MS-CSDS animals compared to that in Std-Con rats. This reduction might be attributed to either feeding or metabolic outcomes of stress exposure [36]. Dyslipidemia, oxidative stress, and insulin resistance, as parts of allostatic load [15], could underlie many of the stress-associated metabolic challenges. In our study, dyslipidemia manifested by higher plasma TC concentrations among the animals exposed to CSDS. In humans, stress has been shown to increase serum TC levels [37]. However, in animals, circulating TC is mainly variable in response to chronic stress, with a decrease or increase, or even without any significant changes [38, 39]. The elevated TC content might be related to the inhibition of its degradation in the liver due to oxidative stress [40]. Earlier studies have reported enhanced oxidative stress secondary to acute and chronic psychological stress in both animals and humans, yet in different contexts [24, 41–43]. According to this, our findings implied that chronic adversity during early or adult life induced systemic oxidative damage in animals as is evident from raised plasma MDA level along with a considerable decline in the GSH content and catalase activity. The intensified production and release of glucocorticoids may modulate ROS generation and mitochondrial calcium homeostasis, through which oxidative damage increases [9, 44]. Furthermore, it is noteworthy that hyperactivity of two other major physiological stress systems, namely the immune-inflammatory system and the autonomic nervous system, is accompanied by oxidative stress [45–47]. Accordingly, the increased oxidative damage in response to either early or adult life adversity in this study may be also a consequence of increased activity of two other stress systems. In the present paper, the elevated circulating corticosterone levels were associated with increased fasting plasma insulin and HOMA2-IR and unchanged plasma fasting glucose among MS-CSDS animals, which could reflect insulin resistance. In line with this association, elevated levels of circulating glucocorticoid have been reported to influence the insulin signalling cascade at the level of insulin receptor [48] and insulin-sensitive glucose transporter 4 (GLUT4) functionality [49, 50]. The increased β cells workload, in the face of insulin resistance [51], could in long term lead to β cell dysfunction and diagnostic hyperglycaemia [52].

Furthermore, stress-related oxidative damage of pancreas in the context of diabetes risk was suggested by López-López et al. [53]. Oxidative damage has been revealed to suppress insulin production in isolated β-cell of pancreas [54]. Accordingly, in the present study we found that the combination of early and adult life adversity could significantly increase the pancreatic enzyme CAT activities in the MS-CSDS animals. On the other hand, adult life adversity significantly raised GSH content among Std-CSDS and MS-CSDS rats, although this increase was more robust among MS-CSDS rats than Std-CSDS group. Despite the increased compensatory antioxidant mechanisms in MS-CSDS rats, pancreatic MDA level, as a marker of lipid peroxidation, elevated in these animals. This is in accordance with the result reported by Sokolova and collegues who showed increased SOD antioxidant enzyme level in the insulin-producing islet by injecting alloxan to mimic oxidative stress-induced onset of diabetes. Thus, it would make sense that augmented pancreatic oxidative stress in MS-CSDS animals exceeded the increased compensatory antioxidant mechanisms, as evidenced by excessive peroxidation of cell membrane lipids. On the other hand, inflammasomes, such as NLRP3 (as mediators of different forms of oxidative stress), induce activation of caspase-1 and subsequent inflammatory cytokine interleukin IL-1β [55]. Based on the central role of IL-1β in the destruction of pancreatic β cells [56–58], it could be of particular relevance in relation to oxidative stress-mediated β cell damage and development of diabetes in response to stress. The findings herein shed light on the fact that early life adversity significantly raised the level of pancreatic IL-1β in response to later chronic social challenges in adulthood. In this context, progressive oxidative and inflammatory cellular damage of pancreas in response to early and adult life adversity synergism might adversely affect β cell function, initiating a related risk of diabetic pathology.

Conclusion

Overall, our study provided evidence that accumulative influences of stress experiences induced corticosterone hypersecretion, insulin resistance, and hyperinsulinemia followed by oxidative and inflammatory damages to the pancreas. Therefore, it would be expected that early adverse experiences unavoidably predispose pancreatic β cells to develop prediabetes in response to later adversity.

Limitations of study

According to the limitations of this work, there are some suggestions to be considered as follows: investigating the expression levels of glucocorticoid receptors in the brain areas involved in the negative feedback of the HPA axis activity, the levels of sympatoadrenal neurohormones, systemic IL-1β levels and the adrenal histological assessments.

Supplementary Information

Below is the link to the electronic supplementary material.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.