Chronic psychosocial stress in male mice causes an up-regulation of scavenger receptor class B type 1 protein in the adrenal glands

Abstract

Mice exposed to chronic subordinate colony housing (CSC, 19 days) show an exaggerated adrenal corticosterone response to an acute heterotypic stressor (elevated platform (EPF), 5 min) despite no difference from EPF-exposed single-housed control (SHC) mice in corticotropin (ACTH) secretion. In the present study, we asked the question whether this CSC-induced increase in adrenal capability to produce and secrete corticosterone is paralleled by an enhanced adrenal availability and/or mobilization capacity of the corticosterone precursor molecule cholesterol. Employing oil-red staining and western blot analysis we revealed comparable relative density of cortical lipid droplets and relative protein expression of hormone-sensitive lipase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and low-density lipoprotein receptor (LDL-R) between CSC and SHC mice. However, relative protein expression of the scavenger receptor class B type 1 (SR-BI) was increased following CSC exposure. Moreover, analysis of plasma high-density lipoprotein-cholesterol (HDL-C) and LDL-cholesterol (LDL-C) revealed increased LDL-C levels in CSC mice. Together with the pronounced increase in adrenal weight, evidently mediated by hyperplasia of adrenocortical cells, these data strongly indicate an enhanced adrenal availability of and capacity to mobilize cholesterol in chronic psychosocially-stressed mice, contributing to their increased in vivo corticosterone response during acute heterotypic stressor exposure.

• Adrenal cortex
• chronic subordinate colony housing
• cholesterol
• chronic psychosocial stress
• HPA axis
• scavenger receptor class B type 1

Introduction

Repeated or chronic exposure to a homotypic stressor is known to result in adaptation of the hypothalamus–pituitary–adrenal (HPA) axis (Aguilera, 1994; Wood et al., 2010). Moreover, considering the deleterious effects of a prolonged elevation of plasma glucocorticoid levels on physical and mental health (McEwen, 1998), the faster the HPA axis habituates to repeated or chronic innocuous homotypic stressors the better it is for the long-term health of the organism (Kudielka et al., 2006; Sasse et al., 2008). However, a phenomenon called sensitization enables the adapted organism to even show an increased HPA axis response to a subsequent heterotypic and possibly life-threatening challenge. To date the mechanisms underlying these adaptive/sensitizing processes are at least partly understood at higher HPA axis levels, i.e. the paraventricular nucleus of the hypothalamus and the anterior pituitary gland (Aguilera, 1994; Wood et al., 2010).

Interestingly, we recently provided evidence for such processes to occur also at the level of the adrenal glands, at least during acute heterotypic stressor exposure subsequent to chronic psychosocial stress (Uschold-Schmidt et al., 2012). Male mice exposed to the chronic subordinate colony housing (CSC, 19 days) paradigm, an adequate and clinically relevant mouse model of chronic psychosocial stress (Reber & Neumann, 2008; Reber et al., 2007, 2008; Slattery et al., 2012), are characterized by unaffected basal morning plasma corticosterone concentrations and an exaggerated corticosterone response to acute heterotypic stressor exposure (elevated platform (EPF) exposure, 5 min), when compared with single-housed control (SHC) mice, despite the corticotropin (ACTH) secretory response to EPF exposure being comparable in the CSC and SHC groups (Uschold-Schmidt et al., 2012). Thus, it seems that CSC mice have an increased adrenocortical capacity to produce and secrete corticosterone in response to acute heterotypic stressor exposure. Similar findings were described previously also in rats; exposure to one single session of immobilization for 2 h resulted in an increased corticosterone response to an elevated plus-maze (EPM) exposure 7 days later, despite plasma ACTH levels that were comparable to respective EPM-exposed control rats (Belda et al., 2008).

In line with these findings, we have further revealed a comparable or even increased relative protein and/or mRNA expression of the ACTH receptor (melanocortin-2-receptor; Mc2r), the Mc2r accessory protein (MRAP) and key steroidogenic enzymes following 19 days of CSC. Together with the CSC-induced increase in adrenal weight, these data indicate how the increased capability of CSC mice to produce and secrete corticosterone during acute heterotypic stressors might be mediated, at least partly (Uschold-Schmidt et al., 2012).

In the present study, we tested the hypothesis that the adrenal availability and/or mobilization capacity of the corticosterone precursor molecule cholesterol (Azhar & Reaven, 2002; Brown & Goldstein, 1986; Gwynne & Strauss, 1982; Kraemer, 2007) is also increased by 19 days of CSC, further adding to the enhanced in vivo corticosterone response to an acute heterotypic stressor following chronic psychosocial stress.

In an adrenocortical cell, cholesterol can be derived from at least four different sources, with endogenous de novo synthesis from acetyl coenzyme A (acetyl CoA) via the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Rosol et al., 2001) being one of them. In addition, cholesterol can also be obtained via receptor-mediated endocytotic uptake of low-density lipoproteins (LDL). Here, the intact lipoprotein is internalized from the blood by the LDL receptor (LDL-R) and then degraded in lysosomes to cholesteryl esters. These cholesteryl esters are hydrolyzed by lysosomal acid lipase, resulting in free cholesterol for steroidogenesis (Kovanen et al., 1979). However, the most prominent amounts of adrenal lipoprotein-derived cholesteryl esters are obtained through the “selective” uptake via the scavenger receptor class B type 1 (SR-BI). Cholesterol-rich high-density lipoproteins (HDL) and to a lesser extent also low density lipoproteins (LDL) bind to the SR-BI, located in the plasma membrane, subsequently leading to the release of cholesteryl esters directly into the cell without internalizing the lipoprotein particle itself (Gwynne & Strauss, 1982; Krieger, 1999; Reaven et al., 1996; Williams et al., 1999). These cholesteryl esters are then hydrolyzed by the hormone-sensitive lipase (HSL) to free cholesterol (Krieger, 1999). Finally, free cholesterol can also be obtained by HSL-mediated hydrolyzation of cholesteryl esters, stored in lipid droplets within the cytoplasm of mostly zona fasciculata cells (Kraemer, 2007).

Thus, to assess if CSC increases the availability and/or mobilization capacity of adrenal cholesterol, we analyzed in the present study the amount of cortical lipid droplets and protein expression levels of HSL, HMG-CoA reductase, LDL-R and SR-BI in left and right adrenal tissue of CSC and SHC mice. Moreover, we analyzed the availability of the main ligands for the LDL-R and the SR-BI, namely HDL cholesterol (HDL-C) and LDL cholesterol (LDL-C), in the plasma of CSC and SHC mice.

Methods

Animals

Male C57BL/6 mice (Charles River, Sulzfeld, Germany) with an age of 36–45 days and a body weight of 19–22 g (experimental mice) were individually housed in standard polycarbonate mouse cages (16 cm × 22 cm × 14 cm) for 1 week before the CSC paradigm started. The male offspring (weighing 30–35 g) of high anxiety-related behavior female mice (kindly provided by Prof. Dr. R. Landgraf, Max Planck Institute of Psychiatry in Munich) and C57BL/6 male mice (Charles River, Sulzfeld, Germany) were used as resident/dominant animals. All mice were kept under standard laboratory conditions (12 h light/dark cycle, lights on at 06:00 h, 22 °C, 60% humidity) and had free access to tap water and standard mouse diet. All experimental protocols were approved by the Committee on Animal Health and Care of the local government, and conformed to international guidelines on the ethical use of animals. All efforts were made to minimize the number of animals used and their suffering.

Experimental procedures

Two sets of experimental mice were either chronically stressed by 19-day exposure to the CSC paradigm or housed singly for control (SHC).

One set of CSC and SHC mice was used to assess the effects of CSC on the number of adrenal cells per given area, and the relative amount of adrenal lipid droplets, the other set of CSC and SHC mice was used to measure adrenal protein expression of HSL, HMG-CoA reductase, LDL-R, SR-BI, as well as plasma LDL-C and HDL-C levels. Therefore, SHC and CSC mice were killed in the morning of day 20 between 08:00 and 10:00 h by decapitation under CO₂ narcosis and trunk blood was collected. Afterward, left and right adrenal glands were removed, pruned of fat, weighed and treated according to the respective readout parameter as described below.

Chronic subordinate colony housing

The CSC paradigm was conducted as described previously (Reber & Neumann, 2008; Reber et al., 2007, 2008; Schmidt et al., 2010; Singewald et al., 2009; Veenema et al., 2008). Briefly, 1 week after arrival, experimental mice were weighed and in a weight-matched manner assigned to the SHC or the CSC group. SHC mice remained undisturbed in their home cage except for change of bedding once a week. CSC mice were housed in groups of four together with a dominant male for 19 consecutive days, in order to induce a chronic stressful situation. To avoid habituation during the chronic stressor exposure, each dominant male was replaced by a novel one at days 8 and 15. SHC and CSC mice were again weighed on day 20, immediately before being killed by decapitation as described above.

Determination of adrenal weight

After decapitation on day 20, the left and right adrenal glands of each mouse were removed, pruned of fat and weighed separately. Values represent absolute adrenal weight (mg). In addition, the sum of left and right absolute adrenal weights was calculated for each mouse.

Afterward, the adrenals were used to assess either the number of adrenal cells per given area and the relative amount of adrenal lipid droplets (Set 1), or protein expression of HSL, HMG-CoA reductase, LDL-R and SR-BI (Set 2).

Cryo-sectioning of adrenal tissue

After removal on day 20 of CSC exposure, left and right adrenal glands were pruned of fat, embedded in protective freezing medium (Tissue-Tek, Sakura Finetek Europe, Zoeterwoude, The Netherlands) and stored at −80 °C. Subsequently, for each left and right adrenal series of five 5-µm cryo-sections (containing both adrenal cortex and medulla) were cut using a cryostat (at −20 °C) and then thaw-mounted onto pre-coated slides (SuperFrost Plus; Menzel-Gläser, Braunschweig, Germany).

DAPI staining in adrenal cryo-sections

To assess the effects of CSC on adrenal cell number, nuclear staining was performed in adrenal cryo-sections. Briefly, one series of 5-µm adrenal cryo-sections of each left and right adrenal gland were air-dried for 10 min at room temperature (RT) and then fixed in cold acetone for 15 min at 8 °C. Afterward, the sections were again air-dried before being stained with a solution of the nuclear dye 4,6-diamidino-2-phenylindol (DAPI, Invitrogen, Darmstadt, Germany) for 30 s. Finally, sections were mounted with fluorescence mounting medium (Fluorescent Mounting Medium, Dako, Glostrup, Denmark) and covered with a glass cover slip. The positively stained area per given area [5000 µm²] was assessed three times for each section in the cortex and the medulla in digitized images using Leika QWin V3 Software (Leika Microsystems, Wetzlar, Germany) and averaged per section. In addition, determination of the average nucleus size allowed calculation of the number of nuclei (equivalent to cells) [n] per given area. The results for each zone of two to five adrenal sections per mouse were pooled (individually for left and right adrenals) to provide individual means.

Oil-red staining in adrenal tissue

To assess the effects of CSC on the availability of cortical cholesteryl esters, adrenal lipid droplets were stained with oil-red as previously described (Ramirez-Zacarias et al., 1992). Briefly, one series of 5-µm adrenal cryo-sections of each left and right adrenal gland were fixed in 4% paraformaldehyde for 3 days. Afterward, the sections were washed in distilled water, rinsed in 60% isopropyl alcohol for 5 min and then stained in a freshly filtered oil-red solution (Certistain Oilred O, Merck, Darmstadt, Germany) for 10 min. The sections were then differentiated in 60% isopropyl alcohol and washed again in distilled water. Sections were mounted with glycerine jelly and covered with a glass cover slip. The entire area per section of all lipid droplets [pixels] as well as the cortex area [pixels] containing these lipid droplets (=relative cortical lipid expression per area) were measured in digitized images using Leika FW4000 Software (Leika Microsystems, Wetzlar, Germany). The results for two to five adrenal sections per mouse were pooled (individually for left and right adrenals) to provide individual means.

Western blotting for adrenal protein expression of HSL, LDL-R, SR-BI and HMG-CoA reductase

Left and right adrenal glands were removed on day 20 of CSC, pruned of fat, immediately shock-frozen in liquid nitrogen and stored at −80 °C until assayed. For protein extraction, frozen left and right adrenals were homogenized separately in ethylendiamintetraacetic acid (EDTA) lysis buffer (50 mM EDTA, 250 mM NaCl, 0.5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 0.5% Igepal, 10% Complete Mini Protease Inhibitor (Roche Diagnostics GmbH, Mannheim, Germany)) and total protein concentration was determined using a commercially available detection kit (Bicinchoninic Acid Protein Assay Kit, Thermo Scientific, Rockford, IL). Western blot analysis was carried out using 20 μg of protein per adrenal. Samples were loaded on sodium dodecyl sulphate polyacrylamide gels (10%) and subsequently transferred on nitrocellulose membranes. The membranes were then blocked for 1 h at RT in 5% BSA (for HSL protein expression) or 5% milk powder (for LDL-R, SR-BI and HMG-CoA reductase), both diluted in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBST, Sigma-Aldrich, Steinheim, Germany), before being probed with primary rabbit anti-HSL (1:1000; Cell Signaling Technology, New England Biolabs GmbH, Frankfurt am Main, Germany), anti-LDL-R (1:500; Abcam, Cambridge, UK), anti-SR-BI (1:1600; Abcam, Cambridge, UK) or anti-HMG-CoA reductase (1:500; Santa Cruz Biotechnology, Inc., Heidelberg, Germany) antibodies overnight at 4 °C. Visualization was performed using horseradish peroxidase-conjugated donkey anti-rabbit antibody (1:3000; GE Healthcare, Freiburg, Germany) followed by ECL Western Blotting Detection Reagents (GE Healthcare, Freiburg, Germany). Immunoblots were digitized using Molecular Imager® ChemiDoc™ XRS+ system and analyzed using Image Lab™ software (Bio Rad Laboratories GmbH, München, Germany). Afterward, each membrane was stripped using Re-Blot Plus Mild Antibody Stripping Solution (Millipore GmbH, Schwalbach, Germany), blocked twice with 5% milk powder in TBST for 5 min and probed with primary rabbit anti-β-tubulin antibody (1:1000, Cell Signaling Technology, New England Biolabs GmbH, Frankfurt am Main, Germany) for 1 h at RT. Visualization and digitization was performed as described above (horseradish peroxidase-conjugated donkey anti-rabbit antibody 1:1000). Bands were detected at ∼82 kDA for HSL, at ∼150 kDA for LDL-R, at ∼76 kDA for SR-BI, at ∼97 kDA for HMG-CoA reductase and at ∼50 kDA for the loading control β-tubulin, as specified by the manufacturers. Expression of all proteins was normalized to the respective β-tubulin protein expression and averaged separately for both left and right adrenal per group.

Determination of plasma HDL-C and LDL-C

On day 20, SHC and CSC mice were rapidly killed by decapitation between 08:00 and 10:00 h. Trunk blood was collected in EDTA-coated tubes (Sarstedt, Nürnbrecht, Germany) on ice and centrifuged at 4 °C (4200 × g, 10 min). Biochemical plasma analysis for HDL-C and LDL-C was performed in the certified Institute for Clinical Chemistry and Laboratory Medicine of the University Hospital Regensburg.

Statistics

For statistical comparisons, the software package SPSS statistics (version 19.0) was used. Data of two experimental groups (SHC versus CSC) were compared using the Student’s t-test. Data of four experimental groups such as adrenal weight, amount of cortical lipid droplets, protein expression of HSL, HMG-CoA reductase, LDL-R and SR-BI (factors CSC and body side) and adrenal cell number (factor CSC and adrenal zone) were compared using a two-way analysis of variance (ANOVA) followed by a post hoc Bonferroni test when appropriate. Data represent the mean + SEM. Significance was taken at p < 0.05.

Results

CSC increases left and right adrenal weight

Absolute weight of both adrenals (combined left and right adrenal weights per mouse) was significantly increased in CSC compared with SHC mice (p < 0.001, Figure 1b). Statistical analysis, considering the factors body side as well as CSC, revealed a significant main effect of both factor body side (F_1,130 = 37.07; p < 0.001) and factor CSC (F_1,130 = 21.74; p < 0.001) with increased weights of left (p < 0.001) and right (p = 0.011) adrenals in CSC compared with SHC mice (Figure 1a). Moreover, the weight of the left adrenal was greater than that of the right adrenal gland in both SHC and CSC mice (for each p < 0.001; Figure 1a).

Figure 1. Effects of chronic subordinate colony housing (CSC) on absolute adrenal weight. Adrenal glands of single-housed control (SHC) and CSC mice were collected on day 20 and weighed. Weights [mg] of left and right adrenal glands, separately and combined, of SHC (n = 34; a/b) and CSC (n = 33; a/b) mice are shown. Symbols indicate significant differences between the groups; within group differences are detailed in results.

SHC;

CSC. Data are mean + SEM. *p < 0.05, ***p < 0.001 versus respective SHC mice (two-way ANOVA, factor CSC and factor body side (a); Student’s t-test (b)).

CSC does not affect the number of adrenal cells per given area

Statistical analysis, considering the factors CSC and adrenal zone, revealed that the number of adrenal cells per given area (Figure 2c) in both the left and right adrenal gland was only dependent on factor adrenal zone (left: F_1,66 = 68.09; p < 0.001; right: F_1,66 = 85.03; p < 0.001; Figure 2a and b), with a greater number of cells per given area in the cortex compared with the medulla in both SHC and CSC mice (for each p < 0.001; Figure 2a and b).

Figure 2. Effects of chronic subordinate colony housing (CSC) on adrenal cell density. Adrenal glands of single-housed control (SHC) (n = 18; a/b) and CSC (n = 17; a/b) mice were collected on day 20. Number of cells [n] per unit area [5000 µm²] was quantified in DAPI-stained sections.

SHC;

CSC. Data are mean + SEM. ###, p < 0.001 versus respective cortex (two-way ANOVA, factor CSC and factor adrenal zone). Representative image of adrenal cryo-section stained with DAPI showing measurement areas (c).

CSC does not affect the relative amount of lipid droplets in the adrenal cortex

Statistical analysis, considering the factors body side (F_1,72 = 0.20; p = 0.653) as well as CSC (F_1,72 = 3.26; p = 0.075), revealed no effects on the relative (per area) amount of lipid droplets (Figure 3c) in the left and right adrenal cortex (Figure 3a). Furthermore, calculation of the sum of relative left and right amount of lipid droplets also revealed no effect of CSC exposure (p = 0.155; Figure 3b).

Figure 3. Effects of chronic subordinate colony housing (CSC) on cortical lipid droplet density. Adrenal glands of single-housed control (SHC) (n = 18; a/b) and CSC (n = 20; a/b) mice were collected on day 20. The area of lipid droplets [pixel] per cortex area [pixel] in oil-red stained sections was determined.

SHC;

CSC. Data are mean + SEM (two-way ANOVA, factor CSC and factor body side (a); Student's t-test (b)). Representative images of cryo-sections stained with oil-red (c).

CSC does not affect relative adrenal protein expression of HSL and HMG-CoA reductase

No effects of factor CSC (HSL: F_1,28 = 1.31; p = 0.262; HMG-CoA reductase: F_1,28 = 0.09; p = 0.759) or factor body side (HSL: F_1,28 = 0.01; p = 0.914; HMG-CoA reductase: F_1,28 = 0.38; p = 0.541) were found on relative (per 20 µg of total protein) protein expression of HSL (Figure 4a and b) or HMG-CoA reductase (Figure 4c and d).

Figure 4. Effects of chronic subordinate colony housing (CSC) on adrenal hormone-sensitive lipase (HSL) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase protein expression. Following decapitation on day 20, the left and right adrenal glands of single-housed control (SHC; n = 8) and CSC (n = 8) mice were collected on day 20. Protein was extracted for determination of HSL (a/b) and HMG-CoA reductase (c/d) protein expression [grey density] normalized to the loading control β-tubulin.

SHC;

CSC. Data are mean + SEM (two-way ANOVA, factor CSC and factor body side). Representative images of bands detected for HSL (∼82 kDA; b) and HMG-CoA reductase (∼97 kDA; d) and respective loading control ß-tubulin (∼50 kDA; b/d) are shown.

CSC does not affect relative adrenal LDL-R protein expression, but increases relative adrenal SR-BI protein expression

Relative (per 20 µg of total protein) LDL-R protein expression in both the left and right adrenal glands was neither affected by factor CSC (F_1,28 = 3.45; p = 0.074) nor by factor body side (F_1,28 = 0.02; p = 0.888) (Figure 5a and b).

Figure 5. Effects of chronic subordinate colony housing (CSC) on adrenal low-density lipoprotein receptor (LDL-R) and scavenger receptor class B type 1 (SR-BI) protein expression. Adrenal glands of single-housed control (SHC; n = 8) and CSC (n = 8) mice were collected on day 20. Protein was extracted for determination of LDL-R (a/b) and SR-BI (c/d) protein expression [grey density] normalized to the loading control β-tubulin (b/d).

SHC;

CSC. Data are mean + SEM. **p < 0.01, ***p < 0.001 versus respective SHC mice (two-way ANOVA, factor CSC and factor body side). Representative images of bands detected for LDL-R (∼150 kDA; b), SR-BI (∼76 kDA; d) and respective loading control β-tubulin (∼50 kDA; b/d) are shown.

In contrast, relative SR-BI protein expression was found to be dependent on factor CSC (F_1,28 = 22.27; p < 0.001; Figure 5c and d). Post hoc analysis revealed a significant increase in relative SR-BI protein expression in both the left (p = 0.008) and right (p = 0.001) adrenal glands in CSC compared with SHC mice.

CSC increases plasma LDL-C, but does not affect plasma HDL-C

While CSC exposure did not affect plasma HDL-C (p = 0.969, Figure 6a) concentrations, plasma LDL-C concentrations were significantly increased in CSC compared with SHC mice (p = 0.003; Figure 6b).

Figure 6. Effects of chronic subordinate colony housing (CSC) on plasma high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C) concentrations. HDL-C [mg/dl] (a) and LDL-C [mg/dl] (b) concentrations were determined on day 20 in trunk blood plasma from SHC (n = 8) and CSC (n = 8) mice.

SHC;

CSC. Data are mean + SEM. **p < 0.01 versus respective SHC mice (Student’s t-test).

Discussion

In the present study, we reveal that the relative amount of cortical lipid droplets as well as relative expression of HSL, HMG-CoA reductase and LDL-R protein is unaffected, whereas the expression of SR-BI protein is enhanced after CSC exposure. Together with the pronounced increase in adrenal weight, which seems to be mediated by cellular hyperplasia, and the increased plasma LDL-C levels, these data strongly indicate an enhanced adrenal availability and mobilization capacity of cholesterol in chronic psychosocially-stressed mice, contributing to their increased in vivo corticosterone response during acute heterotypic stressor exposure.

In agreement with well-documented effects of chronic stressor exposure in general (Retana-Marquez et al., 2003; Schmidt et al., 2007; Westenbroek et al., 2005) and the CSC paradigm in particular (Reber et al., 2007; Uschold-Schmidt et al., 2012), CSC exposure in the present study resulted in a pronounced increase in adrenal weight, confirming the reliability of CSC as chronic psychosocial stress model. As there was a comparable number of cells per given area in SHC and CSC adrenals, this indicates the CSC-induced increase in adrenal weight to be mediated by hyperplasia rather than hypertrophy, resulting in an overall increased number of functional adrenal cells in CSC compared with SHC mice.

Furthermore, oil-red staining in adrenal cryo-sections revealed a comparable relative amount (in relation to the cortex area) of cortical lipid droplets in SHC and CSC mice. Thus, together with the increased number of adrenocortical cells, this indicates an overall enhanced availability of cholesteryl esters from lipid vesicles following chronic psychosocial stressor exposure. Although this seems in contrast to a reduced size of adrenocortical lipid droplets following repeated exposure to combined acoustic and restraint stress in mice (Depke et al., 2008), it might simply be explained by differences in the experimental procedure. Mice in the Depke study were killed immediately following the last 2 h acoustic/restraint stressor exposure, whereas CSC mice in the present study were killed under basal conditions on day 20 of CSC.

Moreover, relative (in relation to the loading control) protein expression of HSL was also not affected by CSC exposure, indicating that metabolism of cholesterol esters to the corticosterone precursor cholesterol is not impaired, or given the increased number of adrenal cells is even enhanced, in adrenal tissue of CSC mice. In addition, relative protein expression of LDL-R was unaffected on day 20 of CSC. Together with the elevated levels of plasma LDL-C in CSC compared with SHC mice and the increased number of adrenal cells following CSC, this indicates that cholesterol derived from endocytotic uptake of blood LDLs is enhanced following CSC, likely contributing as well to the enhanced corticosterone response of CSC mice to acute heterotypic stressors (Uschold-Schmidt et al., 2012).

Interestingly, relative protein expression of adrenal SR-BI was increased following CSC exposure, together with the increased number of adrenal cells indicating a pronounced enhancement of the selective cellular uptake of cholesterol from HDL and LDL following CSC, especially as plasma LDL-C was also increased in CSC compared with SHC mice. As cholesterol taken up by SR-BI represents the major extracellular source for adrenal steroidogenesis in rodents (Gwynne & Strauss, 1982; Kraemer, 2007), this is likely to contribute most to the increased corticosterone response to acute heterotypic stressors of CSC mice (Uschold-Schmidt et al., 2012). Furthermore, relative protein expression of HMG-CoA reductase was comparable between SHC and CSC mice, indicating that endogenous cellular de novo synthesis of cholesterol from acetyl CoA was also not affected, or given the increased number of adrenal cells in CSC mice was overall even increased, following CSC exposure.

Together with our previously reported results of unaffected or even enhanced expression of adrenal Mc2r, MRAP and key steroidogenic enzymes following CSC (Uschold-Schmidt et al., 2012), these data clearly indicate that the adrenal glands of CSC mice overall have greater capacity to produce and secrete corticosterone. This is in line with our recent findings showing that CSC mice respond with higher plasma corticosterone concentrations to acute heterotypic stressor exposure (EPF, 5 min) compared with SHC mice, despite plasma ACTH levels following EPF exposure that were comparable between the CSC and SHC groups (Uschold-Schmidt et al., 2012). Furthermore, these findings clearly indicate that the CSC-induced lack of adrenal corticosterone secretion during in vitro ACTH stimulation (Reber et al., 2007; Uschold-Schmidt et al., 2012) is not due to a general breakdown of the adrenal capability to produce and secrete corticosterone, but is due to a still unknown mechanism, which has to be addressed in future studies. The results rather lead us to the speculation that an additional factor present during acute stressor exposure in vivo, e.g. the sympathetic nervous system (Edwards & Jones, 1987; Ulrich-Lai & Engeland, 2002), supports adrenal ACTH sensitivity or itself acts as a corticosterone secretagogue and, together with the increased capacity of the adrenals to produce corticosterone, leads to the increased glucocorticoid response to acute heterotypic stressors in CSC mice (Uschold-Schmidt et al., 2012).

In summary, the data of the current study indicate an enhanced adrenal availability and mobilization capacity of cholesterol in chronic psychosocially-stressed mice, mainly mediated by an increased adrenal SR-BI expression and increased plasma LDL-C levels, contributing to their increased in vivo corticosterone response during acute heterotypic stressor exposure.

Declaration of interest

The authors thank the German Research Foundation for funding (Research Grant RE 2911/5-1). The authors report no declaration of interest. The authors alone are responsible for the content and writing of the paper.

Acknowledgements

The authors are grateful to N. Grunwald for excellent experimental assistance. We also thank Dr. C. Hellerbrand and B. Czech (Department of Internal Medicine I, University Hospital Regensburg) for their help to measure plasma lipoprotein cholesterol.