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Molecular Endocrinology, doi:10.1210/me.2006-0187
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Molecular Endocrinology 21 (1): 126-137
Copyright © 2007 by The Endocrine Society

Liver X Receptors Regulate Adrenal Steroidogenesis and Hypothalamic-Pituitary-Adrenal Feedback

Maria Nilsson, Thomas M. Stulnig, Chin-Yo Lin, Ai Li Yeo, Peter Nowotny, Edison T. Liu and Knut R. Steffensen

Department of Biosciences and Nutrition (M.N., K.R.S.), Karolinska Institutet, 14157 Huddinge, Sweden; Genome Institute of Singapore (C.-Y.L., A.L.Y., E.T.L.), Singapore 138672; Division of Endocrinology and Metabolism (T.M.S., P.N.), Department of Internal Medicine III, University of Vienna, 1090 Vienna, Austria

Address all correspondence and requests for reprints to: Knut R. Steffensen, Department of Biosciences and Nutrition, Karolinska Institutet, S-14157 Huddinge, Sweden. E-mail: knut.steffensen{at}biosci.ki.se.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The nuclear hormone receptors liver X receptor {alpha} (LXR{alpha}) (NR1H3) and LXRß (NR1H2) are established regulators of cholesterol, lipid, and glucose metabolism and are attractive drug targets for the treatment of diabetes and cardiovascular disease. Adrenal steroid hormones including glucocorticoids and mineralocorticoids are known to interfere with glucose metabolism, insulin signaling, and blood pressure regulation. Here we present genome-wide expression profiles of LXR-responsive genes in both the adrenal and the pituitary gland. LXR activation in cultured adrenal cells inhibited expression of multiple steroidogenic genes and consequently decreased adrenal steroid hormone production. In addition, LXR agonist treatment elevated ACTH mRNA expression and hormone secretion from pituitary cells both in vitro and in vivo. Reduced expression of the glucocortioid-activating enzyme 11ß-hydroxysteroid dehydrogenase 1 in pituitary cells upon LXR activation suggests blunting of the negative feedback of glucocorticoids by LXRs. In conclusion, LXRs independently interfere with the hypothalamic-pituitary-adrenal axis regulation at the level of the pituitary and the adrenal gland.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
METABOLIC SYNDROME IS characterized by obesity and insulin resistance leading to hypertension, dyslipidemia, and glucose intolerance, which can develop into type 2 diabetes (T2D) and provoke cardiovascular disease (CVD). These diseases have become a major health hazard in society and according to the latest World Health Organization estimate, 177 million people suffered from diabetes worldwide in 2000, and this number will increase to at least 300 million by 2025 (http://www.who.int/en). The development of T2D and CVD is most likely of polygenetic origin, and research has focused on understanding regulation of glucose, cholesterol, and lipid metabolism to elucidate their pathophysiology. Several studies indicate a contribution of adrenal steroids for the development of metabolic syndrome. For instance, glucocorticoids have multiple effects on glucose and lipid metabolism, and glucocorticoid excess can elicit visceral obesity and development of metabolic syndrome, but circulating glucocorticoid levels are normal in prevalent forms of obesity (1, 2). Also the main mineralocorticoid, aldosterone, has been reported to interfere with insulin signaling and glucose metabolism (3).

Several nuclear hormone receptors including liver X receptors (LXRs) have been suggested as potential drug targets for the treatment or prevention of T2D and CVD (reviewed in Ref. 4). Liver X receptor (LXR){alpha} (NR1H3) and LXRß (NR1H2) belong to the nuclear hormone receptor superfamily of transcription factors. LXRs are established regulators of cholesterol, lipid, and glucose metabolism. For instance, activation of LXRs regulates the conversion of cholesterol to bile acids, induces lipid/triglyceride biosynthesis, promotes reverse cholesterol transport from peripheral cells to the liver, suppresses gluconeogenesis, and induces influx of glucose from the circulation (reviewed in Refs. 5, 6, 7, 8). Furthermore, LXRß–/– mice, but not LXR{alpha}–/– mice, are glucose intolerant due to impaired insulin secretion from pancreatic ß-cells when challenged with glucose (9). This is in agreement with a study showing that activated LXRs increased glucose-induced insulin secretion in pancreatic ß-cells (10). Intriguingly, the LXRs seem to be involved in energy metabolism by regulating uncoupled oxidative phosphorylation via uncoupling proteins (11, 12). LXRß was reported to up-regulate basal levels of renin whereas LXR{alpha} was involved in cAMP-induced levels of renin (13). These observations indicate that LXRs have important regulatory roles in the maintenance of normal cholesterol, lipid, and glucose metabolism as well as blood pressure homeostasis and could hence be critical factors involved in development of T2D and CVD.

We have recently shown that LXRs regulate the production of the adrenal steroid hormone glucocorticoid and that there are several LXR-responsive genes in adrenal glands (11). Hence, the role(s) of LXRs in adrenals in general, and adrenal hormone production in particular, might be of significant importance to fully understand the effect of LXR signaling in a variety of metabolic processes. Because adrenal glucocorticoid hormone production is under tight control by the hypothalamic-pituitary-adrenal (HPA) axis, we investigated possible roles of LXRs in regulation of the HPA axis including analyses of the molecular mechanisms involved.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To investigate the roles of LXRs in steroidogenesis, we initially analyzed mRNA expression of LXR{alpha} and LXRß in human adrenals, human ovaries, and four different human cell lines; the adrenal cortex H295R carcinoma, the adrenal cortex SW13 adenocarcinoma, the ovary NIH:Ovcar3 carcinoma, and the ovary A2780 carcinoma cells. LXRß was, by far, the dominant LXR paralogue in adrenals, ovaries, and all the investigated cell lines (Fig. 1AGo). Expression of both LXR{alpha} and LXRß was somewhat lower in the cell lines compared with the respective primary tissues except for expression of LXR{alpha} in the adrenal cell lines. To investigate LXR signaling in these cell lines, we treated the four different cell lines with the LXR agonist GW3965 for 0.5 h to 48 h and compared expression profiles at different time points to the dimethylsulfoxide (DMSO)-treated control. Clustering of the LXR-responsive genes showed that the four cell lines grouped in well-defined branches and that each time point branched off compared with the control (supplemental Fig. 1AGo published as supplemental data on The Endocrine Society’s Journals Online website at http). LXR-responsive genes in either of the cell lines at any time point annotated to cholesterol/steroid or lipid metabolism were clustered (supplemental Fig. 1Go, B and C, respectively). These cluster diagrams also showed that there are LXR-responsive genes in all the cell lines; however, the effect of the LXR agonist in the human adrenal H295R cell line was particularly strong. A pronounced change in gene expression (both induction and suppression) due to the treatment was seen on a large set of genes. Expression of many genes was changed as early as after 1–3 h, whereas the largest effect was seen after 24 and 48 h. Together these observations suggest that the optimal effect of activated LXR would be found using the H295R cell line. Thus, to widen the search for LXR target genes in the adrenal cortex, H295R cells were treated with GW3965 for 24 and 48 h, and gene expression profiling was performed using a whole human genome Affymetrix array with 47.000 transcripts. Annotated genes involved in cholesterol/steroid or lipid metabolism with more than 1.5-fold change at both 24 and 48 h were clustered together. A strong induction of expression of several known LXR target genes including ABCA1, ABCG1, and SREBF1 (the latter corresponding to SREBP1) was seen indicating a significant response to the LXR agonist (Fig. 1BGo). Gene expression profiles were verified in H295R cells by quantitative PCR (qPCR). The known LXR target gene ABCA1 was strongly induced by the treatment after 3 h with the strongest induction after 24 and 48 h (Fig. 2AGo). In addition, suppressed expression of genes involved in cholesterol metabolism and steroid hormone production was verified at 10, 24, and 48 h (Fig. 2BGo). The suppressed expression was also observed at the protein level for steroidogenic acute regulatory protein (StAR)(Fig. 2CGo).


Figure 1
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  		Fig. 1. LXR Expression and Genome-Wide Profiling of LXR-Responsive Genes

A, Relative expression of LXRs in adrenals and ovaries. Expression levels of LXRs were quantified in commercially available human RNA from adrenals and ovaries as well as in human cell lines from adrenal cortex (H295R and SW13) and ovaries (Ovcar and A2780). Tissues and cell lines were analyzed in quadruplicate, and the mean relative expression levels of LXR{alpha} and LXRß were compared with the expression of LXR{alpha} in adrenals, the expression of which was set to 1.0 ± SEM. B, H295R cells were treated with DMSO (control) or GW3965 (2 µM). Genes involved in steroid and lipid metabolism, the expression of which is either increased (red) or decreased (blue) at both 24 h and 48 h in response to GW3965 more than 1.5-fold (signal log ratio ± 0.6), are shown.

 

Figure 2
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  		Fig. 2. Verification of LXR-Responsive Genes by qPCR

H295R cells were treated with GW3965 for various time intervals as indicated, and the expression of the LXR target gene ABCA1 (A) and several genes involved in cholesterol metabolism (B) was quantified. The average relative change of expression of three independent experiments for the various time intervals was compared with the expression of the control (0 h = without GW3965), the expression of which was set to 1.0 ± SEM. Expression of the 30-kDa StAR and 42-kDa ß-actin proteins in independent triplicates of protein extract form H295R cells in the absence (DMSO) or presence of GW3965 for 48 h was analyzed (C). DHCR, Dehydroxycholesterol reductase; HMGCS, hydroxy-3-methylglutaryl CoA synthase; HMGCR, hydroxy-3-methylglutaryl CoA reductase; SCARB, scavenger receptor type B; SC4MOL, sterol C4-methyloxidase; LIPE, lipase, hormone sensitive; NSDHL, NAD(P)-dependent steroid dehydrogenase-like; FDFT1, farnesyl-diphosphate farnesyl transferase 1; ABCA1, ATP binding cassette, subfamily A, member 1.

 
To further investigate whether the effect of the LXR agonist was directly on transcription or via mRNA stability, we pretreated the H295R cell line with GW3965 and analyzed the effect of GW3965 in presence of actinomycin D, an inhibitor of transcription, during 48 h as indicated in Fig. 3AGo. Pretreatment with the LXR agonist suppressed expression of cytochrome P450, family 11, subfamily A, polypeptide 1 (Cyp11A1), StAR, steroidogenic factor 1 (SF1) (NR5A1), low-density lipoprotein receptor (LDL-R), and acetyl-coenzyme A acetyltransferase 2 (ACAT2) (Fig. 3Go, B–F, at 0 h). Because an alteration of mRNA stability is still possible, but no mRNA synthesis is allowed in the presence of actinomycin D, we related the effect of the LXR agonist at each time point to the respective controls, which were set to 100%. We did not observe any additional significant change of trend toward additional increased or decreased mRNA levels in the presence of actinomycin D. This suggests that the LXR effect is purely transcriptional rather than an effect on the mRNA stability. Second, the effect of GW3965 was analyzed in the presence and absence of the protein synthesis inhibitor cycloheximide (CHX). CHX did not interfere with the GW3965-mediated induction of expression of ABCA1, verifying a direct transcriptional effect of LXR on the ABCA1 gene (Fig. 3GGo). However, GW3965 could not suppress expression of Cyp11A1, StAR, SF1, LDL-R, ACAT2, and ACTH receptor (ACTH-R) in the presence of CHX (Fig. 3HGo). Thus, LXR-mediated suppression is dependent on new protein synthesis indicating an indirect transcriptional regulatory effect of LXR on these genes.


Figure 3
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  		Fig. 3. mRNA Stability and Protein Synthesis Analysis

A, H295R cells were pretreated with DMSO (Control) or 2 µM GW3965 for 16 h. Growth medium was changed, and all cells were incubated with actinomycin D with DMSO or 2 µM GW3965 from 0–48 h as indicated. B–F, The relative expression levels of the GW3965 treatment in the presence of actinomycin D were compared with its control which at all time points were set to 100%. Error bars indicate ±SEM of the qPCRs of the time points where each was run in triplicate. G and H, H295R cells were pretreated with either DMSO, 1.25 mM (CHX1), or 12.5 mM (CHX2) for 3 h and subsequently treated for 24 h with either DMSO or GW3965 (2 µM). The relative expression levels of ABCA1 (G) and Cyp11A1, StAR, SF1, LDL-R, ACAT2, and ACTH-R (H) in response to the treatments were compared with DMSO treatment alone ± SEM. *, P < 0.001. Ctr., Control.

 
The effect of knocking down expression of the LXRs using small interfering RNA (siRNA) oligos was performed in the H295R cells. siRNA targeting either LXR{alpha} or LXRß suppressed mRNA expression by 50% and 70%, respectively (Fig. 4Go). When cells were transfected with siRNA targeting both LXRs, the mRNA expression of Cyp11A1, StAR and SF1 were induced, whereas expression of ABCA1 was not affected by knocking down expression of LXRs. This suggests that LXR is involved in basal expression levels of Cyp11A1, StAR, and SF1, but not ABCA1 in these cells.


Figure 4
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  		Fig. 4. The LXRs Regulate Basal Expression Levels of Steroidogenic Genes

H295R cells were transfected with siRNA oligos targeting LXR{alpha}, LXRß, or luciferase (control) and mRNA levels analyzed by qPCR. The relative fold expression levels in response to siRNA oligos targeting LXR{alpha}, LXRß (LXR{alpha} and LXRß levels), or both LXRs (ABCA1, Cyp11A1, StAR, and SF1 levels) were compared with control treatment alone, which were set to 1.0 ± SEM. *, P < 0.05; **, P < 0.01. Ctr., Control.

 
Interestingly, a large set of genes suppressed by the LXR agonist included genes involved in cholesterol metabolism and steroid hormone production. Therefore we further investigated whether LXR agonists also affected steroid hormone production. There was a clear trend toward a general suppression of steroid hormone production that was statistically significant for the more prevalent steroid hormone precursors including pregnenolone, dehydroepiandrosterone, and androstendione after treatment of the H295R cells with GW3965 (Table 1Go). Previous observations (11) suggested that LXR agonist treatment in vivo under certain conditions leads to increased plasma glucocorticoid concentrations, suggesting that activation of LXR induces expression of steroidogenic genes, whereas in this study LXR activation suppresses steroid hormone production in isolated adrenal cells. Hence, we hypothesized that LXR interferes with HPA axis regulation not only at the adrenal level but also at the pituitary level. To address this, we investigated the role of LXR on ACTH production. LXR agonists (GW3965, T0901317) induced ACTH production in the AtT-20 pituitary cell line (Fig. 5AGo). In addition, in vivo treatment of mice with an LXR agonist significantly elevated ACTH plasma concentrations. This induction was LXR specific because no effect of the LXR agonist was observed in LXR{alpha}ß–/– mice although the knockout mice exhibited higher basal ACTH concentrations (Fig. 5BGo). These data are in accordance with our previous in vivo observations where plasma glucocorticoid levels were induced by the LXR agonist and LXR{alpha}ß–/– mice show higher basal levels of ‘glucocorticoids.


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  		Table 1. LXR Reduces Adrenal Steroid Hormone Production

 

Figure 5
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  		Fig. 5. LXR Induces ACTH Levels

A, The AtT-20 pituitary cell line was treated with DMSO (control), GW3965 (2 µM), or T0901317 (1 µM) (n = 6 in each treatment), as described in Materials and Methods. Medium was collected and ACTH levels were analyzed. B, Serum from wild-type C57BL/6J (n = 4) and LXR{alpha}ß–/– (n = 5) mice, T0901317-treated, and controls were collected and ACTH levels were measured. T, T0901317.

 
Gene expression analysis revealed several LXR target genes involved in various biological and biochemical processes including the known LXR target genes ABCA1 and SREBP1c in the AtT-20 pituitary cell line (Fig. 6AGo and supplemental Fig. 2Go published as supplemental data on The Endocrine Society’s Journals Online website). 11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) converts inactive glucocorticoids to active glucocorticoids, thereby fulfilling an important role in the regulation of the pituitary feedback to glucocorticoids (14). Expression of 11ß-HSD1 in the AtT-20 pituitary cell line was significantly suppressed whereas expression of ACTH was induced in response to the LXR agonist treatment (Fig. 6AGo). Thus, down-regulation of pituitary 11ß-HSD1 activity might interfere with the negative feedback of adrenal glucocorticoids, thereby enhancing pituitary ACTH production. To test this hypothesis, we analyzed gene expression levels in C57Bl/6 mice after 7 d of administration of the GW3965 LXR agonist. As expected, known LXR target genes were induced by the LXR agonist. Expression of SREBP1c was induced more than 500% in liver whereas SREBP1c and ABCA1 were induced 800% and 300% in the pituitary gland, respectively (Fig. 6BGo), suggesting that the treatment was effective. Expression of 11ß-HSD1 was reduced by 30% whereas expression of ACTH was induced 200% in the pituitary gland. Moreover, expression of SREBP1c and ABCA1 were significantly reduced, expression of 11ß-HSD1 induced whereas no difference was observed in expression of ACTH in LXR{alpha}ß–/– mice compared with wild-type mice (Fig. 6CGo).


Figure 6
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  		Fig. 6. LXR Controls Expression of Target Genes in the Pituitary Gland

Cells or tissue was isolated and RNA was extracted for quantification of expression. A, The AtT-20 pituitary cell line was treated with DMSO (control), GW3965 (2 µM), or T0901317 (1 µM) (n = 6 in each treatment). B, GW3965 or DMSO was administered and after 7 d liver and pituitary gland were isolated for RNA extraction (n = 7 in each group). C, Basal expression levels were analyzed in wild-type C57Bl/6 mice compared with LXR{alpha}ß–/– mice (n = 6 in each group). Expression levels in the treated groups or in LXR{alpha}ß–/– mice are compared with the controls, which are set as 100% ± SEM. *, P < 0.02; **, P < 0.001. C, Control; SREBP, sterol regulatory element-binding protein; T, T0901317; WT, wild type; SREBP1c, sterol regulatory element binding protein 1c.

 
Increased ACTH levels activate the ACTH-R, which signals through G protein-coupled receptors leading to increased levels of cAMP (15). Treatment of H295R cells with forskolin, which activates adenylyl cyclase and increases intracellular cAMP levels, induced expression of several steroidogenic genes, but not ABCA1 (Fig. 7Go). Forskolin also induced these genes in the presence of GW3965, thereby overriding the suppressed gene-regulatory effect of GW3965 alone. This suggests that in conditions with increased levels of cAMP, as expected with increased ACTH levels, the effect of LXR signaling is blunted in the adrenal cortex. Hence, activation of LXR acts differently depending on whether the effect is locally in the adrenals or also involves activation in the pituitary gland (Fig. 8Go).


Figure 7
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  		Fig. 7. Activation of the cAMP Signaling Pathway Inhibits the Gene-Regulatory Effects of LXR

Cells were transfected with either DMSO + ethanol (Control), GW3965 (2 µM), 25 µM forskolin, or both GW3965 and forskolin for 48 h in triplicate. The relative expression levels of ABCA1, Cyp11A1, StAR, SF1, LDL-R, ACAT2, and ACTH-R in response to the treatments were compared with Control treatment alone ± SEM. The lowest P value from the analyzed genes within the treatment group was used to indicate the statistical significance of the treatment.

 

Figure 8
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  		Fig. 8. Proposed Model of LXR Signaling in the HPA Axis

In the adrenal gland, activation of LXR inhibits expression of several steroidogenic genes including SF1. Furthermore, export of cholesterol is induced via ABCA1 and influx of cholesterol is suppressed via LDL-R. In the pituitary gland, activation of LXR suppresses expression of 11ß-HSD1, thereby reducing the amount of active glucocorticoids (active GC) inside the pituitary gland. Consequently, the negative feedback of active GC on ACTH production is blunted leading to higher levels of ACTH. Higher levels of ACTH from the pituitary gland override the inhibitory effect of activated LXR in the adrenal gland. Thus, candidate drugs targeting LXR could give dramatically different effects on steroid hormone production depending on whether the drug enters the central nervous system or not.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
There is a relatively high expression of LXRß in all three layers of the adrenal cortex, i.e. the zona glomerulosa, fasiculata, and reticularis as early as embryonic d 15.5 post coitus in mice embryos (16). We have recently reported several adrenal LXR-responsive genes in mice and increased in vivo production of glucocorticoids by activated LXRs (11). These observations suggest a role of the LXRs in adrenal steroidogenesis. Here we show that both LXR{alpha} and LXRß are expressed in the steroidogenic adrenals glands with higher expression of LXRß. Both human adrenal and human ovary cell lines showed many LXR-responsive genes involved in lipid metabolism and steroid hormone production. Particularly the adrenal cortex H295R cell line showed many LXR-responsive genes. Expression of several genes involved in multiple steps of steroid hormone biosynthesis was suppressed by treatment with an LXR agonist including Cyp11A1, LDL-R (low density lipoprotein receptor), ACTH-R, ACAT2, StAR, and SF1. The LDL-R plays a predominant role in providing cholesterol for adrenal steroidogenesis (17). StAR transports steroids across the mitochondrial membrane in adrenals and is rate limiting for steroid hormone production in the adrenal gland by determining the availability of cholesterol for steroid hormone production (18). SF1 induces transcription of adrenal steroidogenic enzymes including Cyp11A1 (19). Cyp11A1 converts cholesterol to pregnenolone and is the first and rate-limiting step of adrenal steroidogenesis (19). ACTH-R in the adrenal cortex binds pituitary ACTH, which induces steroid production (20), and its expression is controlled by SF1 (21). Moreover, production of several steroid hormones and steroid hormone precursors was decreased in adrenal H295R cells by LXR agonist treatment to a very similar extent, indicating the inhibition was mainly due to down-regulation of the initial enzymatic step.

In adrenals, a vast amount of cholesterol is introduced into the steroidogenic pathway, thereby making cholesterol homeostasis a highly dynamic equilibrium. As in other cells, cholesterol homeostasis depends on cholesterol biosynthesis [regulated by hydroxy-3-methylglutaryl CoA reductase (HMGCR)], uptake (LDL-R), and export (ABCA1). Here we suggest that LXRs perform important roles in all aspects controlling adrenal steroidogenesis: first, LXRs sense oxysterol intermediates at the beginning of this most dynamic adrenal cholesterol-metabolizing pathway. Second, introduction of cholesterol into the steroidogenic pathway is consequently inhibited by down-regulation of key factors including StAR and SF1, furthermore suppressing expression of steroidogenic genes such as ACTH-R and CYP11A1. Third, because reduced steroidogenesis greatly diminishes adrenal cholesterol demands, activated LXRs down-regulate cholesterol provision by inducing ABCA1 and repressing uptake/biosynthesis via LDL-R and HMGCR.

Knockdown of expression of the LXRs in the H295R cell line induced basal mRNA levels of the key steroidogenic genes including Cyp11A1, StAR, and SF1 supporting that LXRs normally suppress expression of these genes. We were not able to completely knock down expression of LXRs in the H295R cells. Even at this reduced level of LXR, the GW3965 agonist still had an effect, so we could not completely block the LXR effect—especially the GW3965-induced effect in these cells. Moreover, overexpression of LXR{alpha} or LXRß did not enhance the effect of the LXR agonist (results not shown) suggesting that LXR signaling is activated solely by the levels of available agonist. Notably, 22R-hydroxycholesterol and 20S-hydroxycholesterol are intermediates during Cyp11A1 action as intermediate products of the conversion of cholesterol to pregnenolone and potent LXR agonists (22). Induced steroid hormone production via cholesterol to pregnenolone increases the levels of these oxysterols, subsequently activating LXRs, which then suppress expression of steroidogenesis at multiple levels. Hence, the LXRs might provide negative feedback mechanisms of steroid hormone production in the adrenal gland.

We have shown that activation of LXRs in vivo lead to increased plasma concentrations of corticosterone in mice (11). Because LXR agonist treatment of cultured adrenal cells down-regulated steroidogenesis, we hypothesized that LXRs not only affect adrenal steroid hormone production but also interfere with HPA axis regulation at the pituitary level. This was particularly evident because the increase in plasma glucocorticoids by LXR agonist treatment in vivo was accompanied by a considerable (3-fold) up-regulation of the ACTH receptor in the adrenals (11). The increased ACTH plasma concentrations in mice with activated LXRs shown in this study support a role of LXRs in the HPA axis at the hypothalamic or pituitary level. Several LXR-responsive genes were found in the mouse AtT-20 pituitary cell line, and these cells also showed a significant LXR-dependent increase in ACTH production. Furthermore, we have previously shown that activated LXRs decreased the expression and activity of 11ß-HSD1 in adipocytes (23). 11ß-HSD1 is critically involved in the feedback regulation of the HPA axis as shown by experiments in 11ß-HSD1–/– animals (14). 11ß-HSD1 catalyzes local activation of inactive glucocorticoid hormones. We show here that LXR agonists suppress expression of 11ß-HSD1 in the AtT20 pituitary cell line as well as in vivo in the pituitary gland. Hence, decreased expression of 11ß-HSD1 in the pituitary gland by LXR agonist treatment could result in reduced local concentrations of active glucocorticoid that blunts the negative feedback at the pituitary level. We also observed induced expression of ACTH both in mice and in the AtT20 cell line, which would be a logical consequence of reduced expression of 11ß-HSD1. When cAMP signaling pathways were activated in the H295R cell line, the effects of the LXR agonist were abolished, suggesting that the LXR effects on the pituitary gland override the local effect observed in the adrenals. Hence, LXR signaling might lead to different effects on the HPA axis, depending on where in the axis LXR is activated (Fig. 8Go).

The increased levels of glucocorticoid seen in LXR{alpha}ß–/– mice were not supported by decreased expression of 11ß-HSD1 and increased expression of ACTH. Expression of 11ß-HSD1 was induced in LXR{alpha}ß–/– mice compared with wild-type mice whereas no difference in expression of ACTH was observed. These results suggest additional LXR effects on the HPA axis. We observed a large reduction in expression levels of both SREBP1c and ABCA1 in the pituitary gland of LXR{alpha}ß–/– mice, suggesting that both lipid biosynthesis and lipid transport are impaired. These and other differences, yet to be resolved, could be involved in the various molecular mechanisms underlying the altered glucocorticoid levels observed between wild-type and LXR{alpha}ß–/– mice. It was recently described that treatment with another LXR agonist (T1317) induces expression of StAR in mice, as well as in the Y1 mouse and H295R adrenal cell lines (24) in contrast to our observations. The LXR effect in the human H295R cell line was lower than in the mouse Y1 cell line and, furthermore, cell culture medium contained serum in our experiments. The latter was an important difference because we showed that factors inducing cAMP signaling pathways (such as hormones in serum) interfere with LXR signaling, being plausible explanations for the difference. Nevertheless, these findings warrant further studies to completely understand the effect of LXR on steroidogenesis.

Adrenal steroid hormones are involved in a plethora of endocrine signaling pathways. Glucocorticoids play a major role in many metabolic processes, and high levels of glucocorticoids dispose for increased risk of developing the metabolic syndrome (25). Hence, the role(s) of LXR in regulation of glucocorticoid production might influence several processes underlying the development of the metabolic syndrome, T2D, and CVD. Although much more work remains in order to fully understand the role(s) of LXRs in the HPA axis, we suggest that pharmacologically targeting LXRs at specific sites in the HPA axis could yield a quite different outcome. Our observations suggest that the in vivo effects would reduce adrenal steroid hormone production if it does not enter the central nervous system whereas the opposite effect could be expected if the drug crosses the blood-brain barrier and activates LXRs in the hypothalamus and/or the pituitary gland. As more studies report that adrenal steroids, particularly the glucocorticoids, contribute to the development of the metabolic syndrome, the role(s) of LXR signaling in this pathway could be yet another approach to intercept the development of metabolic disorders.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animal Care and Treatment
The generation of LXR knockout mice has been described elsewhere (26, 27). The LXR knockout mice of mixed 129/Sv and C57BL/6J background used in these experiments were backcrossed with C57BL/6J mice for 10 generations to obtain LXR knockout mice with a C57BL/6J wild-type background. Mice were housed under a 12-h light, 12-h dark cycle and had free access to water and a diet based on a low-fat standard rodent diet (R36; Lactamin AB, Vadstena, Sweden); the mice were maintained in the animal care facility at the Karolinska Institute, Karolinska University Hospital Animal Center (Huddinge, Sweden). Experimental protocols used for animal studies were approved by the local ethics committee for animal experiments, and the guidelines for the care and use of laboratory animals were followed. All wild-type mice were of C57BL/6J background and age matched to the LXR{alpha}ß–/– mice used in the experiments.

For serum ACTH measurements (Fig. 5Go) and RNA expression analysis (Fig. 6Go) mice were fed experimental diets based on standard low-fat chow mixed with vehicle alone or supplemented with 0.025% (wt/wt) of the synthetic LXR agonist T0901317 (23, 28) or GW3965. Wild-type vs. LXR{alpha}ß–/– mice or wild-type controls vs. wild-type LXR agonist-treated mice were assigned to each experimental group as indicated in the figures. Duration of the LXR agonist treatment was 7 d. Mice were trained by exposure to the killing procedure previously to the actual day of death to minimize stress reactions. Mice were quickly killed by CO2 and subsequent cervical dislocation. Mice were alternately killed from different groups to introduce similar conditions within groups. Blood plasma was collected on ice while tissues were snap-frozen in liquid nitrogen and kept at –80 C until isolation of RNA.

Cell Lines, Transfections, RNA Silencing, and Treatments
The human adrenal cortex carcinoma H295R [growth medium; DMEM-HAM F12 (1:1) with glutamin (catalog no. 11039-021), 2.5% Nu-Serum, and 1% ITS+ (BD Biosciences, Bedford MA)], human adrenal cortex adenocarcinoma SW13 [growth medium; DMEM-HAM F12 (1:1) with glutamine catalog no. 11039-021, 10% fetal bovine serum (FBS)], human ovary carcinoma NIH:Ovcar3 [growth medium RPMI 1640 with glutamine (catalog no. 21875–034), 20% FBS], human ovary carcinoma cells A2780 [growth medium; RPMI 1640 with glutamine (catalog no. 21875–034), 10% FBS] and mouse pituitary tumor cells AtT-20 [growth medium; HAM F10 (catalog no. 22390, 2 mM glutamine, 15% horse serum, 2.5% FBS] were maintained in a 5% CO2 humidified atmosphere at 37 C. All media are from Life Technologies, Inc. (Carlsbad, CA), and all cell lines were grown in the presence of penicillin and streptomycin (50 units/ml and 50 µg/ml, respectively). H295R cells (3 x 104) were seeded in 96-well plates, and siRNA was transiently transfected using Dharmafect 1 (Dharmacon, Lafayette, CO) according to protocol. Briefly, cells were transfected in growth medium for 4 d and RNA was isolated. siGENOME SMRT pools (Dharmacon) were used to knock down expression of LXR{alpha} (M-003413-01) and LXRß (M-003412-01). Luciferase GL3 Duplex siRNA oligo (Dharmacon; D-001400-01-05) was used as nontargeting control. In the actinomycin D experiment cells were pretreated with either DMSO or GW3965 for 16 h, and subsequently all samples were treated with 1 µg/ml actinomycin D (Sigma-Aldrich Inc., St. Louis, MO; catalog no. A9415) for various time points as indicated in Fig. 3Go. In the CHX experiment, cells were pretreated with either DMSO or CHX, either 1.25 mM or 12.5 mM, (Sigma-Aldrich Inc.; catalog no. C4859) for 3 h and subsequently treated for 24 h with either DMSO or GW3965 before harvesting the cells. All treatments with the GW3965 LXR agonist were done at 2 µM concentrations. GW3965, CHX, and actinomycin D were dissolved in DMSO.

RNA Extraction, cDNA Synthesis, and qPCR Analysis of mRNA Expression Levels
RNA from commercially available human adrenal tissue and human ovary tissue were purchased (Ambion, Austin, TX) and RNA from four human cell lines as well as from tissue was isolated using RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s protocol. The concentration and quality of the purified total RNA were determined spectrophotometrically at OD260 nm and by the OD260/280 ratio, respectively. Synthesis of single-stranded cDNA was carried out on 0.5 µg RNA using iScript cDNA synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA) following standard protocol. PCR primers were designed using Primer Express Software version 2.0, a program specially provided for primer design using ABI qPCR machines. All primer pairs span intron-exon boundaries, and the sequences are shown in supplemental Table 1Go published as supplemental data on The Endocrine Society’s Journals Online web site. qPCR assay on the basis of SYBR Green I technology was performed with ABI 7500 fast qPCR system (Applied Biosystems, Foster City, CA). For each pair of primers, a dissociation curve analysis was conducted to validate the specificity of the PCR amplification. Primer concentrations of 100 nM were used in all qPCR analyses. We calculated relative changes employing the comparative method using 18S as the reference gene and controls as calibrators as indicated in the figures.

RNA Genome-Wide Profiling
RNA was isolated using RNeasy Mini Kit (QIAGEN, GmbH), and the quality was determined using the Nano 6000 Chip in the Bioanalyzer from Agilent, Inc. (Palo Alto, CA). RNA (500 ng) was used for one-cycle cDNA synthesis according to the Affymetrix manual (www.affymetrix.com). Hybridization of final targets to the human genome U133 Plus 2.0 Array representing 47,000 transcripts and mouse genome 430A 2.0 array representing 14,000 transcripts followed by probing and scanning were performed according to the Affymetrix manual. The scanned output files were analyzed using Affymetrix Micro Array Suite Version 5.0 software. The gene chips were globally scaled to an average intensity of 500 U to allow comparison of gene expression between samples. Comparisons were made using Affymetrix Data Mining Tool Version 3.0. Signal log ratio of each comparison was used to denote expression as increased (I) or suppressed (S) according to the Affymetrix statistical algorithm. The significance analysis of microarrays (SAM) method was used as a statistical approach to analyze the data, focusing on changed genes identified by Micro Array Suite 5.0. SAM calculates a score for each gene on the basis of the change in expression relative to the SD of all measurements. For genes with scores greater than an adjustable threshold, SAM uses permutations of the repeated measurements to estimate the percentage of genes identified by chance, i.e. the false discovery rate. The q value for each gene represents the probability that it is falsely called significantly changed. Sorting of genes into functional biological groups was done using NetAffx Analysis Center (www.affymetrix.com) and GeneOntologyConsortium (www.geneontology.org). The Gene Expression Omnibus accession number for the Affymetrix data is GPL2999.

Hormone Measurements
The H295R and AtT-20 cell lines were maintained as described above. Cells were pretreated with DMSO, GW3965, or the T0901317 LXR agonist for 24 h. Media were replaced, and cells were incubated another 24 h with the same compounds. Media were collected for analysis. Serum from mice was collected as described above. Adrenal steroids from the H295R cell line were analyzed as reported previously (29) by gas chromatography-mass spectrometry in selected ion monitoring mode and after electric fragmentation using an HP 5973 MSD (Hewlett Packard, Palo Alto, CA) equipped with a 25-m CB5 fused-silica column using individual standardization for each steroid. Sensitivity was 1 pg per injection. ACTH was determined in blood plasma in duplicates by a commercial RIA kit (Phoenix Pharmaceuticals, Inc., Mountain View, CA) according to the manufacturer’s instructions.

Western Blot Protein Analysis
Protein extracts were subjected to 12% SDS-PAGE and transferred to Hybond-P membrane (Amersham Pharmacia Biotech, Piscataway, NJ) by standard techniques. The membrane was blocked for 1 h at room temperature in PBS-T (PBS + 0.05% Tween 20) complemented with 5% milk powder. The membrane was incubated with the primary antibody for 1 h at room temperature in PBS-T with 5% milk powder and subsequently washed three times 10 min in PBS-T. The primary antibody was detected using horseradish peroxidase-linked antirabbit secondary antibody with the enhanced chemiluminescence plus detection kit (Amersham Pharmacia Biotech). Primary antibodies were: StAR (Affinity Bioreagents, Golden CO; catalog no. PA1–560) and the internal control ß-actin (Sigma-Aldrich, Inc.; catalog no. A-5441).


   ACKNOWLEDGMENTS
 
GW 3965 was kindly provided by Tim Willson at GlaxoSmithKline.


   FOOTNOTES
 
This work was supported by grants from the Norwegian Research Council (to K.R.S.) and the Center of Molecular Medicine, a basic research institute within the companies of the Austrian Academy of Sciences and by the Austrian Science Fund; P16788 and P18776 (to T.M.S.).

Disclosure Summary: None of the authors has anything to declare.

First Published Online September 14, 2006

Abbreviations: ACAT2, Acetyl-coenzyme A acetyltransferase 2; ACTH-R, ACTH receptor; CHX, cycloheximide; CVD, cardiovascular disease; Cyp11A1, cytochrome P450, family 11, subfamily A, polypeptide 1; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; 11ß-HSD1, 11ß-hydroxysteroid dehydrogenase type 1; LDL-R; low density lipoprotein receptor; LXR, liver X receptor; PBS-T, PBS + 0.05% Tween 20; qPCR, quantitative PCR; SAM, significance analysis of microarrays; SF1, steroidogenic factor 1; siRNA, small interfering RNA; StAR, steroidogenic acute regulatory protein; T2D, type 2 diabetes.

Received for publication May 4, 2006. Accepted for publication September 5, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Bjorntorp P, Rosmond R 2000 Obesity and cortisol. Nutrition 16:924–936[CrossRef][Medline]
  2. Wajchenberg BL 2000 Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome. Endocr Rev 21:697–738[Abstract/Free Full Text]
  3. Giacchetti G, Sechi LA, Rilli S, Carey RM 2005 The renin-angiotensin-aldosterone system, glucose metabolism and diabetes. Trends Endocrinol Metab 16:120–126[CrossRef][Medline]
  4. Shulman AI, Mangelsdorf DJ 2005 Retinoid x receptor heterodimers in the metabolic syndrome. N Engl J Med 353:604–615[Free Full Text]
  5. Handschin C, Meyer UA 2005 Regulatory network of lipid-sensing nuclear receptors: roles for CAR, PXR, LXR, and FXR. Arch Biochem Biophys 433:387–396[CrossRef][Medline]
  6. Li AC, Glass CK 2004 PPAR- and LXR-dependent pathways controlling lipid metabolism and the development of atherosclerosis. J Lipid Res 45:2161–2173[Abstract/Free Full Text]
  7. Steffensen KR, Gustafsson JA 2006 Liver X receptors: new drug targets to treat type 2 diabetes? Future Lipidol 1:181–189[CrossRef]
  8. Kalaany NY, Mangelsdorf DJ 2006 LXRs and FXR: the yin and yang of cholesterol and fat metabolism. Annu Rev Physiol 68:159–191[CrossRef][Medline]
  9. Gerin I, Dolinsky VW, Shackman JG, Kennedy RT, Chiang SH, Burant CF, Steffensen KR, Gustafsson JA, Macdougald OA 2005 LXRß is required for adipocyte growth, glucose homeostasis, and ß cell function. J Biol Chem 280:23024–23031[Abstract/Free Full Text]
  10. Efanov AM, Sewing S, Bokvist K, Gromada J 2004 Liver X receptor activation stimulates insulin secretion via modulation of glucose and lipid metabolism in pancreatic ß-cells. Diabetes 53(Suppl 3):S75–S78
  11. Steffensen KR, Neo SY, Stulnig TM, Vega VB, Rahman SS, Schuster GU, Gustafsson JA, Liu ET 2004 Genome-wide expression profiling; a panel of mouse tissues discloses novel biological functions of liver X receptors in adrenals. J Mol Endocrinol 33:609–622[Abstract/Free Full Text]
  12. Kalaany NY, Gauthier KC, Zavacki AM, Mammen PP, Kitazume T, Peterson JA, Horton JD, Garry DJ, Bianco AC, Mangelsdorf DJ 2005 LXRs regulate the balance between fat storage and oxidation. Cell Metab 1:231–244[CrossRef][Medline]
  13. Morello F, de Boer RA, Steffensen KR, Gnecchi M, Chisholm JW, Boomsma F, Anderson LM, Lawn RM, Gustafsson JK, Lopez-Ilasaca M, Pratt RE, Dzau VJ 2005 Liver X receptors {alpha} and ß regulate renin expression in vivo. J Clin Invest 115:1913–1922[CrossRef][Medline]
  14. Harris HJ, Kotelevtsev Y, Mullins JJ, Seckl JR, Holmes MC 2001 Intracellular regeneration of glucocorticoids by 11ß-hydroxysteroid dehydrogenase (11ß-HSD)-1 plays a key role in regulation of the hypothalamic-pituitary-adrenal axis: analysis of 11ß-HSD-1-deficient mice. Endocrinology 142:114–120[Abstract/Free Full Text]
  15. Enyeart JJ 2005 Biochemical and ionic signaling mechanisms for ACTH-stimulated cortisol production. Vitam Horm 70:265–279[CrossRef][Medline]
  16. Annicotte JS, Schoonjans K, Auwerx J 2004 Expression of the liver X receptor {alpha} and ß in embryonic and adult mice. Anat Rec A Discov Mol Cell Evol Biol 277:312–316[CrossRef][Medline]
  17. Martin G, Pilon A, Albert C, Valle M, Hum DW, Fruchart JC, Najib J, Clavey V, Staels B 1999 Comparison of expression and regulation of the high-density lipoprotein receptor SR-BI and the low-density lipoprotein receptor in human adrenocortical carcinoma NCI-H295 cells. Eur J Biochem 261:481–491[Medline]
  18. Jefcoate C 2002 High-flux mitochondrial cholesterol trafficking, a specialized function of the adrenal cortex. J Clin Invest 110:881–890[CrossRef][Medline]
  19. Hu MC, Hsu HJ, Guo IC, Chung BC 2004 Function of Cyp11a1 in animal models. Mol Cell Endocrinol 215:95–100[CrossRef][Medline]
  20. Cooper MS, Stewart PM 2005 Diagnosis and treatment of ACTH deficiency. Rev Endocr Metab Disord 6:47–54[CrossRef][Medline]
  21. Frigeri C, Tsao J, Czerwinski W, Schimmer BP 2000 Impaired steroidogenic factor 1 (NR5A1) activity in mutant Y1 mouse adrenocortical tumor cells. Mol Endocrinol 14:535–544[Abstract/Free Full Text]
  22. Janowski BA, Willy PJ, Devi TR, Falck JR, Mangelsdorf DJ 1996 An oxysterol signalling pathway mediated by the nuclear receptor LXR {alpha}. Nature 383:728–731[CrossRef][Medline]
  23. Stulnig TM, Oppermann U, Steffensen KR, Schuster GU, Gustafsson JA 2002 Liver X receptors downregulate 11ß-hydroxysteroid dehydrogenase type 1 expression and activity. Diabetes 51:2426–2433[Abstract/Free Full Text]
  24. Cummins CL, Volle DH, Zhang Y, McDonald JG, Sion B, Lefrancois-Martinez AM, Caira F, Veyssiere G, Mangelsdorf DJ, Lobaccaro JM 2006 Liver X receptors regulate adrenal cholesterol balance. J Clin Invest 116:1902–1912[CrossRef][Medline]
  25. Salehi M, Ferenczi A, Zumoff B 2005 Obesity and cortisol status. Horm Metab Res 37:193–197[CrossRef][Medline]
  26. Alberti S, Schuster G, Parini P, Feltkamp D, Diczfalusy U, Rudling M, Angelin B, Bjorkhem I, Pettersson S, Gustafsson JA 2001 Hepatic cholesterol metabolism and resistance to dietary cholesterol in LXRß-deficient mice. J Clin Invest 107:565–573[Medline]
  27. Schuster GU, Parini P, Wang L, Alberti S, Steffensen KR, Hansson GK, Angelin B, Gustafsson JA 2002 Accumulation of foam cells in liver X receptor-deficient mice. Circulation 106:1147–1153
  28. Repa JJ, Turley SD, Lobaccaro JA, Medina J, Li L, Lustig K, Shan B, Heyman RA, Dietschy JM, Mangelsdorf DJ 2000 Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers. Science 289:1524–1549[Abstract/Free Full Text]
  29. Vierhapper H, Nowotny P, Waldhausl W 1986 Stimulation of gonadal steroid synthesis by chronic excess of adrenocorticotropin in patients with adrenocortical insufficiency. J Clin Invest 77:1063–107010[Medline]

NURSA Molecule Pages Link:

Nuclear Receptors:   LXRβ  |  LXRα
Ligands:   T0901317



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