Selenium Protects Mouse Hypothalamic Cells from Glucocorticoid-Induced Endoplasmic Reticulum Stress Vulnerability and Insulin Signaling Impairment

Abstract

The use of glucocorticoid medications is known to cause metabolic side effects such as overeating, excess weight gain, and insulin resistance. The hypothalamus, a central regulator of feeding behavior and energy expenditure, is highly responsive to glucocorticoids, and it has been proposed that it plays a role in glucocorticoid-induced metabolic defects. Glucocorticoids can alter the expression and activity of antioxidant enzymes and promote the accumulation of reactive oxygen species. Recent evidence indicates that selenium can counter the effects of glucocorticoids, and selenium is critical for proper hypothalamic function. This study sought to determine whether selenium is capable of protecting hypothalamic cells from dysfunction caused by glucocorticoid exposure. We treated mHypoE-44 mouse hypothalamic cells with corticosterone to study the effects on cellular physiology and the involvement of selenium. We found that corticosterone administration rendered cells more vulnerable to endoplasmic reticulum stress and the subsequent impairment of insulin signaling. Supplementing the cell culture media with additional selenium alleviated endoplasmic reticulum stress and promoted insulin signaling. These findings implicate a protective role of selenium against chronic glucocorticoid-induced hypothalamic dysfunction.

Keywords: corticosterone; endoplasmic reticulum stress; glucocorticoid; hypothalamus; insulin; selenium.

Figure 1

Corticosterone exposure for 7 days makes mHypoE-44 cells more vulnerable to endoplasmic reticulum stress. (a) Experimental design: cells were exposed to corticosterone (CORT) or vehicle (VEH; ethanol, EtOH) for 7 days, and tunicamycin was added for the last 21 h prior to protein harvest. (b) Pre-exposure to CORT increased the endoplasmic reticulum (ER) stress response induced by tunicamycin, measured as (C/EBP Homologous Protein) CHOP protein levels (two-way ANOVA: interaction: F(1,12) = 42.7, p < 0.0001; Tukey’s post-hoc: VEH/tunicamycin vs. CORT/tunicamycin, **** p < 0.0001; n = 4 for all groups). All values shown are mean ± standard error of the mean.

Figure 2

Endoplasmic reticulum stress impairs insulin signaling in mHypoE-44 cells, an effect amplified by pre-exposure to corticosterone. (a) Experimental design: Panel b—Endoplasmic reticulum (ER) stress was induced by applying tunicamycin for 21 h, and insulin (INS) was added for the last 30 min prior to cell lysis/protein extraction. Panel c: the INS challenge was also performed after 7 days of corticosterone (CORT) application. Panel d: 7 days of CORT application, followed by tunicamycin and the INS challenge. (b) ER stress induction blunted the response to INS (two-way ANOVA: interaction: F(1,12) = 5.5, p = 0.04, Tunicamycin: F(1,12) = 74.1, p < 0.0001; Tukey’s post-hoc: VEH/INS vs. Tunicamycin/INS, ** p = 0.004; n = 4 for all groups). (c) CORT exposure for 7 days slightly reduced the response to INS (two-way ANOVA: CORT: F(1,12) = 4.8, p = 0.049; Tukey’s: VEH/Saline vs. VEH/INS *** p = 0.0005, CORT/Saline vs. CORT/INS *** p = 0.0004; n = 4 for all groups). (d) Pre-exposure to CORT worsened the INS signaling impairment caused by ER stress induction (two-way ANOVA: interaction: F(1,12) = 5.4, p = 0.04; Tukey’s: VEH/INS vs. CORT/INS, * p = 0.02; n = 4 for all groups). All values shown are mean ± standard error of the mean.

Figure 3

Supplementation with selenium has a restorative effect on endoplasmic reticulum stress and insulin signaling in corticosterone-exposed mHypoE-44 cells. (a) Experimental design: panel b: cells were treated with selenium (Se) supplementation (500 nM Se in media) in conjunction with either corticosterone (CORT) or vehicle (VEH; ethanol, EtOH) exposure, followed by endoplasmic reticulum (ER) stress induction using tunicamycin or addition of VEH (dimethyl sulfoxide, DMSO); panel c: cells were treated with Se supplementation in conjunction with either CORT or VEH, then all cells were exposed to tunicamycin, followed by either insulin (INS) or VEH (phosphate-buffered saline, PBS). (b) Culturing cells in media containing 500 nM Se prevented CORT from decreasing the potentiation of ER stress (two-way ANOVA: interaction: F(1,12) = 2.0, p = 0.2; Tukey’s: VEH/tunicamycin vs. CORT/tunicamycin: p = 0.07; n = 4 for all groups). (c) Supplementation with Se prevented CORT from worsening the impairment of INS signaling caused by ER stress (two-way ANOVA: CORT: F(1,12) = 8.9, p = 0.1; Tukey’s: VEH/INS vs. CORT/INS, p = 0.9; n = 4 for all groups). All values shown are mean ± standard error of the mean.

Figure 4

Proposed model of the ability of selenium to protect insulin signaling from being impaired by chronic corticosterone exposure in mHypoE-44 hypothalamic cells. Upon binding insulin, the insulin receptor initiates an intracellular signaling cascade that involves the activation (via phosphorylation) of protein kinase B (AKT). Endoplasmic reticulum (ER) stress limits the phosphorylation of AKT, an effect that is exacerbated by chronic exposure to corticosterone (CORT). Supplementation with additional selenium (Se) is able to prevent CORT from exacerbating ER stress and, subsequently, inhibiting insulin signaling. Red lines highlight the pathological effects of chronic CORT.