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Endocrinology, doi:10.1210/en.2008-0420
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Endocrinology Vol. 149, No. 12 5909-5918
Copyright © 2008 by The Endocrine Society

7-Oxysterols Modulate Glucocorticoid Activity in Adipocytes through Competition for 11β-Hydroxysteroid Dehydrogenase Type

Malgorzata Wamil, Ruth Andrew, Karen E. Chapman, Jonathan Street, Nicholas M. Morton and Jonathan R. Seckl

Endocrinology Unit, Centre for Cardiovascular Science, The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, United Kingdom

Address all correspondence and requests for reprints to: Jonathan R. Seckl, Endocrinology Unit, Centre for Cardiovascular Science, The Queen’s Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. E-mail: j.seckl{at}ed.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Obesity is associated with an increased risk of diabetes type 2, dyslipidemia, and atherosclerosis. These cardiovascular and metabolic abnormalities are exacerbated by excessive dietary fat, particularly cholesterol and its metabolites. High adipose tissue glucocorticoid levels, generated by the intracellular enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), are also implicated in the pathogenesis of obesity, metabolic syndrome, and atherosclerosis. 11β-HSD1 also interconverts the atherogenic oxysterols 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7β-HC). Here, we report that 11β-HSD1 catalyzes the reduction of 7KC to 7β-HC in mature 3T3-L1 and 3T3-F442A adipocytes, leading to cellular accumulation of 7β-HC. Approximately 73% of added 7KC was reduced to 7β-HC within 24 h; this conversion was prevented by selective inhibition of 11β-HSD1. Oxysterol and glucocorticoid conversion by 11β-HSD1 was competitive and occurred with a physiologically relevant IC50 range of 450 nM for 7KC inhibition of glucocorticoid metabolism. Working as an inhibitor of 11β-reductase activity, 7KC decreased the regeneration of active glucocorticoid and limited the process of differentiation of 3T3-L1 preadipocytes. 7KC and 7β-HC did not activate liver X receptor in a transactivation assay, nor did they display intrinsic activation of the glucocorticoid receptor. However, when coincubated with glucocorticoid (10 nM), 7KC repressed, and 7β-HC enhanced, glucocorticoid receptor transcriptional activity. The effect of 7-oxysterols resulted from the modulation of 11β-HSD1 reaction direction, and could be ameliorated by overexpression of hexose 6-phosphate dehydrogenase, which supplies reduced nicotinamide adenine dinucleotide phosphate to 11β-HSD1. Thus, the activity and reaction direction of adipose 11β-HSD1 is altered under conditions of oxysterol excess, and could impact upon the pathophysiology of obesity and its complications.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OBESITY IS A KNOWN risk factor for cardiovascular diseases, yet the molecular link between increased fat mass and atherosclerosis is still unknown. It has been estimated that adipose tissue stores over half of total body cholesterol, mainly as free cholesterol (1), and this proportion increases with adipocyte hypertrophy (2). Adipocytes also remove serum oxidized low-density lipoproteins (oxLDLs) through scavenger receptors (Cluster of Differentiation 36, oxLDL receptor 1, and scavenger receptor class B type I) (1, 3); oxLDL is a major source of proatherogenic 7-oxysterols. This uptake appears beneficial because cholesterol depletion in adipocytes causes perturbation in fatty acid and glucose metabolism (2, 4). Hypertrophied adipocytes from obese rodent models show elevated 3-hydroxy-3-methylglutaryl coenzyme A reductase and low-density lipoprotein receptor mRNAs, suggesting that these cells are cholesterol deficient and have increased cholesterol biosynthesis (2, 5). Thus, adipose tissue is a potential sump to safely store harmful cholesterol metabolites, and these protective qualities appear to be lost during insulin-resistant obesity.

Patients with glucocorticoid excess (Cushing’s syndrome) develop visceral obesity, insulin resistance, diabetes type 2, dyslipidemia, and have an increased risk of cardiovascular mortality. Tissue-specific differences in local cellular glucocorticoid exposure may explain the Cushing’s-like features of idiopathic obesity/metabolic syndrome in the absence of elevated plasma cortisol levels (6). One contention is that the 11-keto-reduction of inert cortisone (11-dehydrocorticosterone in mice and rats) to active cortisol (corticosterone) by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which is elevated specifically in adipose tissue of obese humans and rodents (2, 3, 4), may cause "Cushing’s syndrome of adipose tissue" in idiopathic obesity. Transgenic mice overexpressing 11β-HSD1 selectively in adipose tissue develop many features of the metabolic syndrome, including glucose intolerance, insulin resistance, dyslipidemia, and hypertension (7), whereas 11β-HSD1 null (11β-HSD1–/–) mice are protected from these deleterious effects upon high-fat feeding (8, 9). Crucially, 11β-HSD1–/– mice also exhibit an atheroprotective phenotype, with increased plasma high-density lipoprotein (HDL) cholesterol and lower free fatty acid levels (8, 9). Furthermore, treatment of atherosclerosis-prone apolipoprotein E null mice with an 11β-HSD1 inhibitor attenuates atherogenesis (10). Together, these data suggest a role for elevated adipose 11β-HSD1 levels, driving adipocyte hypertrophy and insulin resistance that, together with consequent downstream vascular and hepatic lipid and cholesterol handling effects, is potentially proatherogenic.

It was reported recently that 11β-HSD1 interconverts 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7β-HC) in the liver (11, 12). 7KC, followed by 7β-HC are the most abundant oxysterols in oxLDL (13), and their concentrations correlate with atherosclerosis risk (14). Although present in nanomolar concentrations in the plasma under physiological conditions (10–100 nM) (15, 16, 17), these 7-oxygenated metabolites are found in micromolar concentrations in human foam cells, atherosclerotic plaques, and plasma of dyslipidemic patients (18). 7-oxysterol levels in adipose tissue have not been reported.

Given the key role of adipose tissue in modulating whole body cholesterol homeostasis (2, 4), we hypothesized that metabolism of these oxysterols by 11β-HSD1 might provide a novel link between obesity, adipose glucocorticoid action, and atherogenesis. We provide evidence that 7-oxysterols are produced enzymatically in adipocytes, regulate 11β-HSD1-dependent conversion of glucocorticoids, and, therefore, signaling by glucocorticoid receptor (GR), and when in excess may inhibit the differentiation of preadipocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
7KC and 7β-HC were obtained from Steraloids (Newport, RI), and [1,2,6,7-3H]4-corticosterone and [1,2,6-3H]3-KC were from Amersham Pharmacia Biotech (Aylesbury, UK). 11-[1,2,6,7-3H]4-Dehydrocorticosterone was prepared as described previously (19). T0901317 and 22(R)-HC were purchased from Cayman Chemicals (Ann Arbor, MI). Solvents were glass-distilled HPLC grade from Rathburn Chemicals (Walkerburn, UK). Other chemicals were purchased from Sigma (Poole, UK). The selective 11β-HSD1 inhibitor (compound 544) was a gift from Merck Chemicals, Darmstadt, Germany.

Cell culture
3T3-F442A cells were propagated in DMEM (Cambrex, Verviers, Belgium) supplemented with 10% new born calf serum, penicillin/streptomycin (50 U/ml and 50 µg/ml, respectively; Invitrogen, Paisley, UK) at 37 C in a humidified atmosphere with 5% CO2. 3T3-F442A cells were differentiated into adipocytes as previously described (20). Briefly, cells were seeded in collagen-coated six-well plates (BD Bioscience, Oxford, UK) and grown to confluence. Two days after confluence, the medium was changed to differentiation medium, comprising DMEM supplemented with 10% fetal bovine serum (FBS), antibiotics (as described previously), and 5 µg/ml insulin. To assess differentiation, 7-d post-confluent cells were stained with Oil Red O and hematoxylin-ammonium water to visualize lipid droplets. 3T3-L1 cells were bought from American Type Culture Collection (Manassas, VA) and differentiated as previously described (20). Briefly, post-confluent cells were incubated for 2 d in DMEM supplemented with 10% FBS, penicillin/streptomycin (as described previously), 0.25 µM dexamethasone (or 11-dehydrocorticosterone and corticosterone where indicated), 200 µM 3-isobutyl-1-methylxanthine (IBMX), and 1 µg/ml insulin, followed by 3 d in medium supplemented with 1 µg/ml insulin, 10% FBS, and antibiotics.

Human embryonic kidney (HEK) 293 cells were maintained at 37 C, 5% CO2 in DMEM supplemented with 10% (vol/vol) FBS, penicillin/streptomycin (as described previously). HEK293(11β-HSD1) cells stably transfected with human 11β-HSD1 were kindly provided by Dr. Scott Webster (Endocrinology Unit, University of Edinburgh, Edinburgh, UK). HEK293(11β-HSD1) cells were maintained identically.

11β-HSD1 activity assays
11β-HSD1 activity in fully differentiated intact 3T3-F442A adipocytes was assayed in 12-well plates in 1 ml serum free medium to which appropriate substrate was added, with radiolabeled tracer. Substrate concentrations used were: 200 nM corticosterone with 5 nM [1,2,6,7-3H]4-corticosterone as tracer (dehydrogenase assay); 200 nM 11-dehydrocorticosterone (11-DHC) with 5 nM 11-[1,2,6,7-3H]4-DHC as tracer (reductase assay); and 500 nM 7KC with 5 nM [1,2,6-3H]3-KC as a tracer. For the enzyme competition assays, two substrates were added in the serum-free medium: one in a constant concentration; and the other in a range of concentrations, 1 nM-100 µM for 7KC and 11-DHC. Due to low solubility of 7-oxysterols in aqueous solutions, higher concentration of 7KC could not be achieved. Stocks of sterols were reconstituted in 100% ethanol and used at 1:1000 or more dilution in culture media. All incubations were in triplicate and performed for the time points indicated in the legend.

For measurement of glucocorticoid metabolism, after incubation, steroids were extracted from 1 ml medium with 3 volumes of ethyl acetate and separated by thin-layer chromatography (TLC) (Merck Chemicals, Darmstadt, Germany) using ethanol-chloroform (8:92) as the mobile phase. 3H-steroids were quantified using a phosphorimager (Fuji FLA-2000; Fujifilm Corp., Tokyo, Japan) and Aida software (Raytek Scientific Ltd., Sheffield, UK). Enzyme activity was expressed as percent conversion of substrate to product after correction for values from control (no cell) incubations.

A well-established protocol for the measurement of glucocorticoid conversion in 11β-HSD1 activity assay was used in the primary work on the development of 7-oxysterol conversion assay. Fully differentiated 3T3-F442A adipocytes were incubated with 7KC diluted 1:1000 from ethanol stock into the medium to give the final concentration. Several methods of extraction of oxysterols from the medium were tested. The recovery from the medium was improved to approximately 50–70% by using 6 volumes of dichloromethane and serum-free medium. Because the recovery of 7-oxysterols from the medium containing serum was very poor, we used serum-free medium in experiments testing the metabolism of 7KC in adipocytes. For measurement of oxysterol metabolism, after incubation, 1 ml medium was pipetted into glass tubes, to which 6 ml dichloromethane was added, and samples were vortexed for 1 min. To study the accumulation of 7-oxysterols in adipocytes (Fig. 1AGo), after incubation with radiolabeled 7KC, whole cellular pellets (without performing further steps of oxysterol extraction from cells) were resuspended in scintillation fluid (Ultima Gold; PerkinElmer Life And Analytical Sciences, Inc., Waltham, MA) and analyzed by the Liquid Scintillation Analyzer (Beckman Coulter, Inc., Fullerton, CA). To extract sterols from cells, cells were washed with PBS, centrifuged for 5 min at 2000 x g, and resuspended in an appropriate volume (200 µl) of lysis buffer [300 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 5% glycerol, and 5% Triton X-100 (pH 7)]. Lysates were incubated for 10 min at 37 C. Oxysterols were extracted with 6 ml ethyl acetate and dissolved in mobile phase (85% acetonitrile, 15% water) for analysis by HPLC with online radioactivity detection (LD509 Berthold absorbance detector; Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany), using a Sun Fire C18 column (15 cm, 4.9 µm particle size; Waters, Macclesfield, UK) at 30 C. Nonradioactive standards were used to optimize resolution conditions with an online dual wavelength absorbance UV detector. The wavelength of UV detection was determined by the structural characteristics of the analytes. After HPLC the area under each peak was integrated using the Xcalibur software (Xcalibur, Inc., Reston, VA) to quantify the percent conversion of radiolabeled 7KC to 7β-HC. The percent conversion in each tissue sample was corrected to blank samples (medium only) included in each experiment. Assays were performed in duplicate. Additional controls used cell-free medium. Tritiated 7β-HC and 7KC were detected at 28 and 32-min retention times, respectively. No additional products of 7KC conversion were observed, either by HPLC or by TLC (data not shown). The complete inhibition of the 7-oxysterol conversion by the selective 11β-HSD1 inhibitor was used as a control for autoxidation during sample preparation. In addition, in samples in which 11β-HSD1 has been heat inactivated, no product of the reaction was present.


Figure 1
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  		FIG. 1. Conversion of 7-oxysterols in fully differentiated 3T3-F442A adipocytes. A, To measure the metabolism of 7KC in differentiated 3T3-F442A adipocytes, cells were incubated with [3H]3-7KC for up to 24 h, and the distribution of radioactivity in the medium and lysed cells was measured by liquid scintillation analysis. Black and open bars represent the percentage of radioactivity detected in the medium and lysed cells, respectively. Data are the means ± SEM of three independent experiments, each performed in duplicate. The effect of time was statistically significant (*, P < 0.05). B, Conversion of [3H]3-7KC to 7β-HC by 3T3-F442A in cell lysates and the medium. 3T3-F442A cells were incubated for 2, 5, or 24 h with 500 nM 7KC and [3H]3-7KC as a tracer before extraction and HPLC analysis. Hatched bars represent 7β-HC extracted from the cell lysate, and open and black bars represent 7KC extracted from the cell lysate and medium, respectively. Data are means ± SEM from three independent experiments, each performed in triplicate. *, P < 0.05 (one-way ANOVA with Bonferroni post hoc tests) for the comparison of extracts in the medium and cellular lysate pellet. C and D, HPLC analysis of oxysterols extracted from 3T3-F442A cells after 24 h incubation with [3H]3-7KC (C) or [3H]3-7KC (D), with the addition of 5 µM 11β-HSD1 inhibitor (compound 544). Two peaks were detected with retention times 28 and 32 min for 7β-HC and 7KC, respectively.

 
GR and liver X receptor (LXR){alpha} activation in cell lines
Transient transfections were performed using Gene Juice Transfection Reagent (Novogen, Berkshire, UK). Cells were seeded at 2 x 105 per well in six-well plates coated with poly-D-lysine (Sigma-Aldrich, Dorset, UK). After overnight incubation, medium was replaced with medium containing 10% charcoal-stripped fetal calf serum, and cells were transfected with a total of 1 µg DNA comprising 0.1 µg pEGFP-rGR (rat) (21, 22), 0.5 µg pLTR-Luc (21), and 0.1 µg pRSV- βgal (internal control) (23) and 0.3 µg H6PDH plasmid (24) or cDNA3.1 (Invitrogen; empty vector) where indicated. pEGFP-rGR is pharmacologically indistinguishable from wild-type GR (21, 22). GR was activated with 10 nM corticosterone or 11-DHC. Alternatively, for LXR{alpha} activation, cells were transfected with 0.1 µg CMX-Gal4-hLXRa, an expression construct encoding a chimeric receptor encoding the LXR ligand binding domain fused to the GAL4 DNA binding domain, 0.5 µg TK-MH100 x 4-Luc luciferase reporter plasmid (25, 26) (a gift from D. J. Mangelsdorf, Southwestern Medical Center, Dallas, TX), and 0.1 µg pRSV-βgal. After overnight incubation, steroids and oxysterols were added for 16 h before harvesting and lysis of cells. LXR{alpha} was activated with 20 µM 22(R)-hydroxycholesterol and 1 µM T0901317 as a positive control (25, 26). Luciferase and β-galactosidase activities in cell lysates were each measured in duplicate as previously described (23). All transfections were performed in triplicate, and values represent mean ± SEM of the ratio of luciferase/β to galactosidase activities.

Real-time RT-PCR
RNA was isolated from cell cultures using TRIZOL reagent (Invitrogen) as previously described (9). Total RNA was quantitated by spectrophotometry at 260 nm. Levels of specific mRNAs were measured by real-time PCR. A total of 1 µg RNA was treated with deoxyribonuclease I (amplification grade; Invitrogen) and transcribed to cDNA using oligo(deoxythymidine)20 and Superscript III (Invitrogen) according to the manufacturer’s instructions. Real-time PCR was performed on a Roche Lightcycler using Master mix (Roche, Burgess Hill, UK) and primer/probe sets from Applied Biosystems (Foster City, CA); Mm00476182_m1 and Mm01138344_m1 to measure 11β-HSD1 and sterol regulatory element-binding protein (SREBP) 1c, respectively. TATA binding protein mRNA levels were used as an internal control.

Statistics
Data are expressed as mean ± SEM and were analyzed using GraphPad Prism software (GraphPad Software Inc., San Diego, CA) (Student’s t test, or one or two-way ANOVA, followed by Newman-Keuls and Bonferroni post hoc tests, as appropriate). Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
3T3-F442A adipocytes accumulate and convert 7KC to 7β-HC
To examine whether 11β-HSD1 in adipocytes metabolizes its 7-ketosterol substrate, we examined conversion of [3H]37KC to 7β-HC in fully differentiated 3T3-F442A adipocytes, which exhibit endogenous 11β-HSD1 reductase activity (20). After 24 h incubation, most of the added radioactivity was detected in the cellular fraction, with less than 17% remaining in the medium (Fig. 1Go, A and B), suggesting sequestration of oxysterols in the adipocytes rather than balanced influx/efflux [the latter is seen in some other cell types (11, 27, 28) and unpublished data, suggesting adipocytes act as a "sink" for 7-oxysterols]. HPLC analysis of oxysterols extracted from the lysed cells showed two peaks, corresponding to 7β-HC and 7KC (Fig. 1CGo), suggesting metabolism was only through 11β-HSD1. After 24 h, approximately 73% of 7KC was converted to 7β-HC (Fig. 1BGo), indicating a predominant 11β-reductase effect on oxysterols as well as glucocorticoid conversion (21). 11β-HSD1 mediation of this reaction was confirmed by the lack of 7KC metabolism in the presence of a selective 11β-HSD1 inhibitor (compound 544) (Fig. 1DGo).

7-Oxysterols compete with glucocorticoids as substrates for 11β-HSD1
To investigate competition between glucocorticoid and oxysterol substrates, both were added together to the medium of 3T3-F442A adipocytes. 7KC (1 nM–100 µM) inhibited the conversion of 11-DHC to corticosterone in a dose-dependent manner, with an "apparent IC50" of 450 nM (95% IC50 0.17–1.18 µM; Fig. 2AGo). This appeared to reflect effects upon metabolism rather than any cell toxicity because MTS assays (4 h incubation in serum-free medium with 200 nM 11-DHC and a range of 7KC concentrations from 0–100 µM) indicated no effect of 7KC on cell viability at the doses used here (data not shown). Similarly, 11-DHC (1 nM–100 µM) inhibited conversion of 7KC to 7βHC (Fig. 2BGo). Although adipocytes show very little 11β-dehydrogenation of corticosterone to 11-DHC, 7KC dose dependently increased 3T3 cell 11β-dehydrogenase activity, albeit this remained at low levels (data not shown).


Figure 2
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  		FIG. 2. Competition between glucocorticoids and 7-oxysterols for 11β-HSD1. A, Dose-dependent inhibition of glucocorticoid conversion by 7KC. 3T3-F442A adipocytes were incubated with 200 nM 11-DHC and 5 nM [3H]4-11-DHC as a tracer with 7KC (1 nM–100 µM) for 4 h. Steroids were extracted from the medium by the addition of ethyl acetate and analyzed by TLC. Data are the mean ± SEM of three independent experiments, each performed in triplicate and are expressed as a percentage of control (no added 7KC). One hundred percent conversion on the graph represents 85% actual conversion in the control. B, Dose-dependent inhibition of oxysterol conversion by 11-DHC analyzed by HPLC. 3T3-F442A adipocytes were incubated with 200 nM 7KC and 5 nM [3H]3-KC as a tracer and 11-DHC (1 nM-100 µM) for 4 h. Oxysterols were extracted from cells and analyzed by HPLC. Data represent the mean ± SEM of three independent experiments, each performed in triplicate and are expressed as percentage of control (no added 11-DHC). One hundred percent conversion on the graph represents 28% actual conversion of 7KC to 7β-HC in the control.

 
7-Oxysterols do not regulate 11β-HSD1 mRNA expression in adipocytes
Liquorice-based inhibitors of 11β-HSD affect transcription of 11β-HSD1 mRNA. However, neither 7KC nor 7β-HC (20 µM) altered 11β-HSD1 mRNA levels when incubated for 24 h with fully differentiated 3T3-F442A (data not shown) or 3T3-L1 adipocytes (Fig. 3AGo), nor did they alter 11β-HSD1 mRNA when present in the medium (1 µM) throughout the 10 d differentiation of 3T3-L1 adipocytes (Fig. 3BGo). This suggests that the effects on 11β-HSD1 activity are not transcriptionally mediated.


Figure 3
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  		FIG. 3. No regulation of 11β-HSD1 mRNA by 7-oxysterols. A, 3T3-L1 adipocytes were incubated for 24 h with 7KC (20 µM), 7β-HC (20 µM), 22(R)-HC (20 µM) and T0901317 (1 µM), rosiglitazone (1 µM; positive control), and a selective11β-HSD1 inhibitor (compound 544; 5 µM), as indicated. Values are 11β-HSD1 mRNA levels relative to TATA-binding protein mRNA, used as an internal standard, and are expressed relative to vehicle-treated cells (arbitrarily set to one). Data are the mean ± SEM of four independent experiments, each performed in duplicate. *, P < 0.05. Similar results were obtained in 3T3-F442A adipocytes (data not shown). B, No regulation of 11β-HSD1 mRNA by 7-oxysterols. 3T3-L1 cells were differentiated according to the standard protocol (including 0.25 µM dexamethasone) with supplementation of 1 µM 7KC (hatched bars), 7βHC (white bars), or vehicle (ethanol; black bars) in the differentiation medium through differentiation (added fresh every second day). RNA was extracted on d 0, 2, 4, and 10 differentiation. Values are the ratio of 11β-HSD1 mRNA levels to TATA binding protein mRNA levels and are means ± SEM from two independent experiments, each performed in triplicate. A significant increase in 11β-HSD1 mRNA was detected on d 4 and 10 differentiation. No significant effect of treatment with 7-oxysterols was found.

 
7-Oxysterols modulate GR-mediated promoter activation
To assess the downstream impact of 11β-HSD1-mediated oxysterol metabolism, we examined GR-mediated transactivation using a HEK293 cell line stably transfected with 11β-HSD1 [HEK293(11β-HSD1)]. HEK293 cells, unlike differentiated 3T3-F442A and 3T3-L1 cells, are readily amenable to transfection. HEK293 cells lack functional GR (29), therefore, HEK293(11β-HSD1) cells were transiently transfected with an expression plasmid encoding GR and a glucocorticoid-sensitive [mouse mammary tumor virus (MMTV)-long terminal repeat (LTR)-luciferase] promoter-reporter construct [HEK293(11β-HSD1) plus GR]. Corticosterone and 11-DHC were equipotent in stimulating reporter gene activity at physiologically relevant (10 nM) concentrations, suggesting rapid reactivation of 11-DHC to corticosterone by 11β-HSD1. This effect was mediated by GR because it was blocked by addition of the GR antagonist RU38486 (RU486) (Fig. 4AGo). Neither 7KC nor 7β-HC (20 µM) alone exhibited any direct agonist activity at GR.


Figure 4
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  		FIG. 4. 7-Oxysterols modulate GR activity of 11-DHC and corticosterone (cort). HEK293(11β-HSD1) (A) or HEK293 cells (B) (without 11β-HSD1) were transfected with EGFP-rGR, MMTV-LTR-luciferase, and pRSV-βgal, and treated with 10 nM glucocorticoid (11-DHC or corticosterone) with the addition of the appropriate 7-oxysterol (20 µM) or RU486 (1 µM). Values are the ratio of luciferase to β-galactosidase activity expressed relative to basal MMTV-LTR activity and are the means ± SEM of four to six independent experiments, each performed in triplicate. *, P < 0.05 for the effect of modulation of glucocorticoid-dependent (i.e. glucocorticoid alone) activation of GR by 7-oxysterols. C, HEK293(11β-HSD1) cells were transfected with GR, MMTV-LTR-luciferase, and pRSV-β, and cotransfected with the H6PDH plasmid and activated with glucocorticoid (10 nM 11-DHC or corticosterone) with or without 7-oxysterols. Results are expressed as the ratio of luciferase to β-galactosidase activity and are the means ± SEM of four separate experiments.

 
To test whether oxysterols affected the ability of 11β-HSD1 to generate active glucocorticoid ligands for GR, HEK293(11β-HSD1) plus GR cells were incubated with 10 nM 11-DHC or corticosterone (chosen to submaximally activate GR) and 7KC or 7βHC (20 µM). Importantly, 7KC (20 µM) inhibited 11-DHC-induced luciferase activity (Fig. 4AGo). Unexpectedly, 7KC also inhibited corticosterone-induced luciferase activity. In contrast, 20 µM 7β-HC enhanced both corticosterone- and 11-DHC-mediated transcriptional activation of the reporter gene (Fig. 4AGo). Similar results were obtained using another glucocorticoid response element-reporter construct driven by the GR-dependent phenylethanolamine N-methyltransferase gene promoter (30) (data not shown). 7-oxysterols did not modulate GR activation when HEK293 cells (not stably transfected with 11β-HSD1) were used (Fig. 4BGo).

Modulation of 11β-HSD1 reaction direction by 7-oxysterols
One mechanism by which 7KC may reduce GR activation by both corticosterone (already active) and inert 11-DHC involves shifting the 11β-HSD1 reaction direction toward 11β-dehydrogenation. To address this we cotransfected HEK293(11β-HSD1) plus GR cells with H6PDH, which drives 11β-HSD1 activity toward oxo-reduction, thus regenerating active corticosterone (24, 29). Consistent with this hypothesis and with previous data in this cell line (29), HEK293(11β-HSD1) plus GR cells exhibited bidirectional glucocorticoid metabolism, at comparable levels (40–50% conversion), when incubated with either 11-DHC (Fig. 5AGo) or corticosterone (Fig. 5BGo), but only 11β-reductase activity when cotransfected with H6PDH (29).


Figure 5
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  		FIG. 5. Modulation of 11β-HSD1 reductase activity (A) and dehydrogenase activity (B) by 7-oxysterols in HEK293(11β-HSD1) cells. Cells were incubated for 16 h with either 10 nM [3H]411-DHC (A) or 10 nM [3H]4 corticosterone (cort) (B), with the addition of 7KC (20 µM), 7β-HC (20 µM), or the selective 11β-HSD1 inhibitor (inhib) (compound 544, 5 µM), as indicated. Values are expressed as percent conversion of substrate to product 11β-reductase direction (A) and 11ββ-dehydrogenase direction (B). Data are the means ± SEM of three independent experiments. *, P < 0.05.

 
7KC (20 µM) inhibited the conversion of 11-DHC to corticosterone (11β-reductase direction; Fig. 5AGo), and promoted conversion of corticosterone to 11-DHC (11β-dehydrogenase direction; Fig. 5BGo) in HEK293(11β-HSD1) cells without H6PDH cotransfection. In contrast, 7β-HC increased the formation of active corticosterone, shifting the 11β-HSD1 reaction direction toward 11β-reduction (Fig. 5Go). Moreover, when H6PDH was overexpressed, 7-oxysterols no longer had effects upon corticosterone-mediated GR transcriptional activity (Fig. 4CGo), and the effects of 7β-HC on 11-DHC transactivation via GR were also blocked, suggesting that these effects were mediated via changes in 11β-HSD1 reaction direction in HEK293 cells. These data suggest that in the presence of submaximal levels of H6PDH, 7KC influences glucocorticoid metabolism by11β-HSD1 toward dehydrogenation, but that, in addition, it antagonizes activation of 11-DHC to active corticosterone by another mechanism, presumably substrate competition.

7-Oxysterols do not activate LXR.
We next examined whether the effects of oxysterols on GR activation downstream of 11β-HSD1 were part of a more generalized "gating" by the enzyme of nuclear receptors. Some oxysterols [e.g. 22(R)-HC] bind to and activate LXRs (31), which may in turn down-regulate adipocyte 11β-HSD1 expression (32). Given the previously reported atheroprotective effects of the 11β-HSD1 inhibition (10), we evaluated the ability of 11β-HSD1 mediated 7-oxysterol metabolism to produce an LXR agonist and modulate LXR-gated activation of genes involved in lipid metabolism. HEK293 cells were transfected with a cDNA encoding a chimeric activator comprising the Gal4 DNA binding domain and the LXR{alpha} ligand binding domain, and a reporter plasmid in which transcription factor activation from Gal4 binding sites is required for luciferase reporter activity (25, 26). Synthetic (Compound T0901317; 250 nM) and physiological [22(R)-HC; 20 µM] LXR agonists activated the reporter by 9- and 5-fold, respectively (Fig. 5AGo). 7KC and 7β-HC were inactive in the trans-activation assay (Fig. 6AGo). The effect of 7KC and 7β-HC upon LXR target genes was also tested in 3T3-F442A adipocytes, which express endogenous LXR (33). Whereas LXR agonists increased endogenous mRNA levels encoding SREBP1c, an LXR target gene in adipose tissue (34, 35). 7KC and 7β-HC were without effect (Fig. 6BGo). In contrast to previous reports (32), the LXR agonists [T0901317-1 µM and 22(R)-HC-20 µM] had no effect on 11β-HSD1 mRNA levels in 3T3-F442A or 3T3-L1 cells (Fig. 3AGo) (data not shown).


Figure 6
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  		FIG. 6. 7-Oxysterols do not activate LXR. A, HEK293(11β-HSD1) cells were transfected with CMX-Gal4-hLXR{alpha}, an expression construct encoding the LXR{alpha} ligand binding domain, TK-MH100 x 4-Luc reporter plasmid and pRSV-βgal (internal control). Transfected cells were incubated with T0901317 (250 nM), 22(R)-HC (20 µM), 7KC (20 µM), or 7β-HC (20 µM). Values represent relative means ± SEM from four independent experiments, each performed in triplicate. *, P < 0.05 for the effect of treatment. B, Fully differentiated 3T3-F442A adipocytes were incubated with 7-oxysterols (20 µM), LXR ligands: 22(R)-HC (20 µM), TO901317 (1 µM), or vehicle for 24 h. Levels of mRNA encoding SEBP1c were measured by real time-PCR. Results represent means ± SEM from three independent experiments, each performed in duplicate. TATA binding protein (TBP) mRNA expression was used as an internal control. *, P < 0.05 for the effect of treatment.

 
7KC inhibits 11β-HSD1-mediated 3T3-L1 preadipocyte differentiation.
Finally, we wished to examine potential functional implications of oxysterol metabolism by 11β-HSD1 in adipocytes. Glucocorticoids are required for the differentiation of 3T3-L1 preadipocytes into the mature adipocyte phenotype (36). 11-DHC (by virtue of its conversion to corticosterone by 11β-HSD1) can substitute for dexamethasone in promoting adipocyte differentiation (31). Given our observations that 7-oxysterols accumulate in adipocytes, we hypothesized that 7KC might inhibit 11-DHC-mediated 3T3-L1 preadipocyte differentiation. Supplementation of the differentiation mixture (containing 250 nM 11-DHC) with 20 µM 7KC decreased the differentiation of 3T3-L1 preadipocytes measured by Oil red O staining by d 10 after addition of supplemented medium (Fig. 7Go). 7KC did not influence differentiation induced by dexamethasone or corticosterone, suggesting that the 7KC-mediated block of differentiation resulted from inhibition of 11β-HSD1 reduction of 11-DHC to corticosterone.


Figure 7
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  		FIG. 7. 7KC inhibits differentiation of 3T3-L1 adipocytes induced by 11-DHC. Representative images of Oil red O stained 3T3-L1 cells 7 d after addition of differentiation mixture: A, Undifferentiated cells (negative control), adipocytes differentiated according to the standard protocol by the addition of B, IBMX, insulin, and dexamethasone; C, IBMX, insulin and 11-DHC (inactive glucocorticoid); D, IBMX, insulin and corticosterone (active glucocorticoid); E, IBMX, insulin, dexamethasone, and 20 µM 7KC; and F, IBMX, insulin, 11-DHC, and 20 µM 7KC. Photos are representative of four independent experiments, each performed in duplicate. Positive results were also obtained when 1 µM 7KC was used. Objective magnification, x40.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A prominent hypothesis underpinning the metabolic and atherosclerotic consequences of obesity is increased 11β-HSD1 in adipose tissue. Here, we show that 7-oxysterols accumulate within adipocytes and that 11β-HSD1 in adipocytes metabolizes these highly proatherogenic oxysterols, predominantly from 7KC to 7β-HC. We describe a novel cross talk between glucocorticoid and oxysterol pathways in that 7KC inhibits glucocorticoid action, whereas 7β-HC enhances glucocorticoid action, and provide functional evidence that this affects preadipocyte differentiation. The effects appear to occur predominantly through substrate competition at the enzyme activity level, and may also include reversal of 11β-HSD1 reaction direction (reductase) if 7KC levels are high and reduced nicotinamide adenine dinucleotide phosphate cofactor levels are limiting within the cell.

Adipocytes accumulated the 7-oxysterol substrate/product of 11β-HSD1, converted 7KC with preferential 11β-reduction activity, as previously described in the liver (11), and sequestered the 7β-HC product. Intact HEK293 cells exhibited balanced influx/efflux of 7-oxysterols rather than sequestration (27, 28). The differences between HEK293 cells and adipocytes highlight the complexity of the metabolism of alternative 11β-HSD1 substrates in various cell systems. The accumulated intracellular 7KC interfered with the ability of 11β-HSD1 to regenerate active glucocorticoids. The relatively low, physiologically relevant IC50 for inhibition of glucocorticoid regeneration by 7KC suggests that such competition may occur in adipose tissue in vivo. In contrast, the much higher IC50 (micromolar) for inhibition of 7KC conversion by 11-DHC is unlikely to have a major impact given the nanomolar physiological levels of circulating 11-DHC (37).

The effects of 7KC were not mediated at the 11β-HSD1 mRNA level. When 7KC levels were high, as might occur in adipocytes as they accumulate and sequester 7-oxysterols, they appeared to promote a change in reaction direction with increased 11β-dehydrogenation of glucocorticoids. In HEK293(11β-HSD1) cells, this change in reaction direction driven by 7KC was overcome by overexpression of H6PDH, believed to be coupled physiologically to 11β-HSD1 in the endoplasmic reticulum and, thus, the major determinant of reaction direction in intact cells. It is unlikely that this situation would occur in mature fully differentiated adipocytes, in which 11β-HSD1 activity is almost exclusively reductase (20, 38). However, the limiting levels of H6PDH in HEK293 cells suggest that where the provision of cofactor is limiting, such as in human preadipocytes (24), accumulation of this oxysterol might attenuate glucocorticoid-mediated functions such as promotion of preadipocyte differentiation, both by substrate competition and perhaps by reaction direction reversal.

Neither 7-oxysterol displayed intrinsic activity toward GR, but both regulated glucocorticoid-dependent GR transcriptional activity. It seems plausible that 7KC competes as a substrate with 11-DHC, consuming endogenous reduced nicotinamide adenine dinucleotide phosphate. 7β-HC potentiated GR mediated transcription, but only when H6PDH was limiting. Thus, 7β-HC promotes the accumulation of active corticosterone in cells in which cofactor is limiting but is unlikely to affect GC action in which H6PDH is abundant, as in mature adipocytes. 7KC attenuated GR transactivation with 11-DHC and corticosterone when H6PDH was limiting, consistent with its role to prevent active glucocorticoid regeneration (HEK293 cells convert corticosterone to 11-DHC, which would not be then reduced back to corticosterone in the presence of 7KC).

11β-HSD1–/– mice have a cardioprotective lipid profile (low low-density lipoprotein, high HDL) (8), and pharmacological inhibition of 11β-HSD1 activity is atheroprotective (10). Although there may be protective effects of 11β-HSD1 deficiency within the vessel wall and on hepatic cholesterol metabolism (9, 39), additionally, the insulin sensitization of 11β-HSD1 deficiency may be atheroprotective by clearing oxLDL from the plasma. Adipocytes undergoing hypertrophy increase the uptake of oxLDL that are high in 7KC because they require high delivery of cholesterol for growing cytoplasmic membranes and storage of triglycerides. Restricted efflux pathway of 7KC and 7β-HC from cells to HDL is completely dependent on the expression of ABCG1 and the presence of lipoproteins (40). In this capacity, atherogenic 7-oxysterols could be trapped in adipocytes as a protective mechanism. In addition, 7KC inhibited differentiation of preadipocytes, extending the previously reported inhibitory effect of oxLDL in general on 3T3-L1 differentiation and proliferation (41). In dyslipidemia, high concentrations of 7KC in adipose tissue might thus inhibit preadipocyte differentiation. Interestingly, the ablation of ABCG1 in adipocytes has reduced obesity (42), presumably through the accumulation of high concentrations of 7-oxysterols and subsequently the cytotoxic effect.

We hypothesize that 7KC accumulation in adipocytes with low levels of 11β-HSD1 or limiting concentrations of H6PDH (such as reported in human preadipocytes or in adipose tissue in lean subjects) might reduce 11β-HSD1-dependent amplification of glucocorticoids and ameliorate some metabolic consequences of obesity. Moreover, 7KC inhibits de novo cholesterol biosynthesis via promoting SREBP cleavage-activating protein-mediated release of SREBP (43), potentially reducing adipocyte lipid accumulation. Indeed, in humans, adipocyte size correlates directly with 11β-HSD1 levels In contrast, 7β-HC may have the opposite effect, and some data suggest it is the more atherogenic of the two sterols (44). Thus, in mature adipocytes with plentiful H6PDH, and especially in obesity when 11β-HSD1 levels are high, 11β-HSD1 is a reductase, regenerating glucocorticoids and accumulating 7β-HC (as seen here in 3T3 cells), which is not only (perhaps) more atherogenic but which, unlike 7KC, does not inhibit de novo cholesterol biosynthesis. Potentially, the balance between these oxysterols, itself determined by 11β-HSD1, may be crucial in modulating glucocorticoid and oxysterol effects in adipose tissue (Fig. 8Go).


Figure 8
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  		FIG. 8. A hypothetical model of the effects of interactions between 7-oxysterol and glucocorticoid substrates of 11β-HSD1 in adipocytes. In cells with low H6PDH levels (HEK293 cells, human preadipocytes) or when 11β-HSD1 levels are low (leanness), 7KC metabolism to 7β-HC consumes cofactor, promoting 11β-HSD1 dehydrogenation, lowering glucocorticoid levels inside cells, attenuating GR activation, and reducing metabolic disease/atherogenic potential. 7KC also reduces de novo cholesterol biosynthesis and may be less atherogenic per se. In contrast, in mature adipocytes (especially in obesity) and differentiated 3T3 cells, 11β-HSD1 and H6PDH levels are high, and the enzyme is a predominant 11β-reductase. This drives regeneration of active glucocorticoids and formation of (putatively) more atherogenic 7β-HC from 7KC. 7β-HC accumulates in adipocytes, facilitates de novo cholesterol biosynthesis, and adds to the metabolic disease burden. CORT, Corticosterone.

 
Studies on purified receptors described 7β-HC as a poor LXR ligand (31). However, sulfated (7KC-3-sulfate) metabolites inhibited transactivation by LXR in vitro (45), suggesting that the activity of these oxysterols for LXR in vivo may be determined by their abundance and modification within the tissue. Moreover, LXR ligands have decreased the expression and activity of 11β-HSD1 in 3T3-L1 cells (32), suggesting that a cross talk between the oxysterol and glucocorticoid pathways may operate also at the transcriptional level. However, 7-oxysterols failed to show any intrinsic activity for LXR{alpha}. Furthermore, no regulation of the 11β-HSD1 mRNA levels of 11β-HSD1 was observed with either LXR ligands or 7-oxysterols in differentiated 3T3-F442A and 3T3-L1 adipocytes after 24 h, in contrast to the previous report (32). The basis for the latter discrepancy is uncertain, but here at least we find no evidence for cross talk between glucocorticoid and LXR{alpha}-mediated pathways in 3T3 cells.

7KC and 7β-HC are abundant oxysterols, known to be cytotoxic at high concentrations and implicated in atherogenesis. Our data have shown that they do not exert direct effects via either GR or LXR. However, they do have the potential to influence glucocorticoid access to GR, by modulation of 11β-HSD1 in adipose tissue. These data could lead to a revision of our understanding of the association between glucocorticoids and atherogenesis. In the future, it will be critical to establish whether a similar regulation of 11β-HSD1 activity occurs in macrophages, especially in foam cells in which oxysterols accumulate to high concentrations, and in which 11β-HSD1 is highly expressed and functionally relevant.


   Acknowledgments
 
We thank Val Lyons for technical assistance and Scott Webster for generously providing human embryonic kidney 293(11β-hydroxysteroid dehydrogenase type 1) cells.


   Footnotes
 
This research was supported by a four-year British Heart Foundation Ph.D., studentship (to M.W.), a Wellcome Trust Research Career Development Fellowship (to N.M.M.), and a Wellcome Trust Programe Grant (to J.R.S. and K.E.C.).

Disclosure Statement: M.W., R.A., K.E.C., J.S., and N.M.M. have nothing to declare. J.R.S. has previously consulted for Incyte, Vitae Pharmaceuticals, Merck, and Ipsen, and is an inventor on European Union WO9707789.

See editorial p. 5907

First Published Online August 28, 2008

Abbreviations: 11-DHC, 11-Dehydrocorticosterone; FBS, fetal bovine serum; GR, glucocorticoid receptor; HDL, high-density lipoprotein; HEK, human embryonic kidney; 7β-HC, 7β-hydroxycholesterol; 11β-HSD1, 11β-hydroxysteroid dehydrogenase type 1; 11β-HSD1–/–, 11β-hydroxysteroid dehydrogenase type 1 null; IBMX, 3-isobutyl-1-methylxanthine; 7KC, 7-ketocholesterol; LTR, long terminal repeat; LXR, liver X receptor; MMTV, mouse mammary tumor virus; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethylphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt; oxLDL, oxidized low-density lipoprotein; RU486, RU38486; SREBP, sterol regulatory element-binding protein; TLC, thin-layer chromatography.

Received March 31, 2008.

Accepted for publication August 19, 2008.


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