Glucoregulation and coping behavior after chronic stress in rats: Sex differences across the lifespan

Abstract

The purpose of the current study was to determine how biological sex shapes behavioral coping and metabolic health across the lifespan after chronic stress. We hypothesized that examining chronic stress-induced behavioral and endocrine outcomes would reveal sex differences in the biological basis of susceptibility. During late adolescence, male and female Sprague-Dawley rats experienced chronic variable stress (CVS). Following completion of CVS, all rats experienced a forced swim test (FST) followed 3 days later by a fasted glucose tolerance test (GTT). The FST was used to determine coping in response to a stressor. Endocrine metabolic function was evaluated in the GTT by measuring glucose and corticosterone, the primary rodent glucocorticoid. Rats then aged to 15 months when the FST and GTT were repeated. In young rats, chronically stressed females exhibited more passive coping and corticosterone release in the FST. Additionally, chronically stressed females had elevated corticosterone and impaired glucose clearance in the GTT. Aging affected all measurements as behavioral and endocrine outcomes were sex specific. Furthermore, regression analysis between hormonal and behavioral responses identified associations depending on sex and stress. Collectively, these data indicate increased female susceptibility to the effects of chronic stress during adolescence. Further, translational investigation of coping style and glucose homeostasis may identify biomarkers for stress-related disorders.

Keywords: Adolescence; Aging; Corticosterone; Forced swim test; Glucose tolerance.

Figure 1:. Experimental design and timeline.

A single cohort of rats was separated into 4 experimental groups consisting of male and female unstressed controls (No CVS, n = 24/sex) and chronically stressed males and females (CVS, n = 36/sex). These groups underwent endocrine and behavioral assessments immediately following CVS. After aging for 13 months, they were re-tested prior to tissue collection.

Figure 2:. Young metabolic measures.

Body weight was measured throughout the course of CVS for both CVS (n = 36/sex) and No CVS (n = 24/sex) controls (A). On the morning of CVS day 14, a baseline blood sample was taken to determine plasma corticosterone (B), plasma triglycerides (C), and plasma cholesterol levels (D). Data are expressed as mean ± SEM. * represents sex differences within stress condition, ^# represents CVS effects within sex, ^† represents sex differences within CVS over time. *,^# p < 0.05, ** p < 0.01, and ****,^†††† p < 0.0001.

Figure 3:. Young FST.

During FST, behavioral coping was assessed as passive (A) or active (B) in No CVS (n = 24/sex) and CVS (n = 36/sex) rats. Blood was taken at 15, 30, 60, and 120 min after the initiation of the 10-min FST. Plasma corticosterone was measured (C) and the integrated total corticosterone response was calculated from the AUC (D). Blood glucose was also measured at each time point (E) and total blood glucose calculated (F). Data are expressed as mean ± SEM. * represents sex differences within stress condition, ^# represents CVS effects within sex (indicated by color for time-dependent measures), ^† represents sex differences within CVS over time. *,^#,^† p < 0.05, **,^##,^†† p < 0.01, ***,^###,^††† p< 0.001, and ****,^†††† p < 0.0001.

Figure 4:. Young GTT.

Baseline blood samples (0 min) were taken before glucose injection (1.5 g/kg, i.p.). Blood was taken at 15, 30, 45, and 120 min post injection in No CVS (n = 24/sex) and CVS (n = 36/sex) rats. At each time point, blood glucose was measured (A) and total blood glucose was calculated from the AUC (B). Plasma corticosterone was also measured (C) and cumulative corticosterone determined from the AUC (D). Data are expressed as mean ± SEM. ↓ represents glucose bolus, * represents sex differences within stress condition, ^# represents CVS effects within sex (indicated by color for time-dependent measures), ^† represents sex differences within CVS over time. *,^#,^† p < 0.05, ^## p < 0.01, ^††† p< 0.001, and ^†††† p < 0.0001.

Figure 5:. Aged baseline and somatic measures.

Following 13 months of aging, body weight (A) and baseline corticosterone (B) were measured. Following euthanasia, body weight-corrected weights of adrenal glands (C), mesenteric white adipose (D), and inguinal white adipose (E) were determined. No CVS: n = 24/sex and CVS: n = 36/sex. Data are expressed as mean ± SEM. * represents sex differences within stress condition. ** p < 0.01 and **** p < 0.0001.

Figure 6:. Aged FST.

During FST, behavioral coping was assessed as passive (A) or active (B) in No CVS (n = 24/sex) and CVS (n = 36/sex) rats. Blood was taken at 15, 30, 60, and 120 min after the initiation of the 10-min FST. Plasma corticosterone was measured (C) and the total corticosterone response was calculated from the AUC (D). Blood glucose was also measured (E) and total blood glucose calculated (F). Data are expressed as mean ± SEM. * represents sex differences within stress condition and ^† represents sex differences within CVS over time. *,^† p < 0.05, **,^†† p < 0.01, *** p< 0.001, and ****,^†††† p < 0.0001.

Figure 7:. Aged GTT.

Baseline blood samples (0 min) were taken before glucose injection (1.5 g/kg, i.p.). Blood was taken at 15, 30, 45, and 120 min post injection in No CVS (n = 24/sex) and CVS (n = 36/sex) rats. At each time point, blood glucose was measured (A) and total blood glucose was calculated from the AUC (B). Plasma corticosterone was also measured (C) and cumulative corticosterone determined from the AUC (D). Data are expressed as mean ± SEM. ↓ represents glucose bolus, ^† represents sex difference within CVS (p < 0.05).

Figure 8:. Regression analysis.

Pearson correlations were run within each group [No CVS (n = 24/sex) and CVS (n = 36/sex)] for all measures taken throughout the study. Significant associations (p < 0.05) are bolded.