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Originally published In Press as doi:10.1074/jbc.M313615200 on February 18, 2004

J. Biol. Chem., Vol. 279, Issue 18, 18415-18424, April 30, 2004
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Rapid Hepatic Metabolism of 7-Ketocholesterol by 11{beta}-Hydroxysteroid Dehydrogenase Type 1

SPECIES-SPECIFIC DIFFERENCES BETWEEN THE RAT, HUMAN, AND HAMSTER ENZYME*

Roberto A. S. Schweizer, Marcel Zürcher, Zoltan Balazs, Bernhard Dick, and Alex Odermatt{ddagger}

From the Division of Nephrology and Hypertension, Department of Clinical Research, University of Berne, 3010 Berne, Switzerland

Received for publication, December 12, 2003 , and in revised form, February 11, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of 11{beta}-hydroxysteroid dehydrogenase type 1 (11{beta}-HSD1) in the local activation of the glucocorticoid receptor by converting inactive 11-ketoglucocorticoids to active 11{beta}-hydroxyglucocorticoids is well established. Currently, 11{beta}-HSD1 is considered a promising target for treatment of obese and diabetic patients. Here, we demonstrate a role of 11{beta}-HSD1 in the metabolism of 7-ketocholesterol (7KC), the major dietary oxysterol. Comparison of recombinant 11{beta}-HSD1, transiently expressed in human embryonic kidney 293 cells, revealed the stereo-specific interconversion of 7KC and 7{beta}-hydroxycholesterol by rat and human 11{beta}-HSD1, whereas the hamster enzyme interconverted 7{alpha}-hydroxycholesterol, 7{beta}-hydroxycholesterol, and 7KC. In contrast to lysates, which efficiently catalyzed both oxidation and reduction, intact cells exclusively reduced 7KC. These findings were confirmed using rat and hamster liver homogenates, intact rat hepatocytes, and intact hamster liver tissue slices. Reduction of 7KC was abolished upon inhibition of 11{beta}-HSD1 by carbenoxolone (CBX) or 2'-hydroxyflavanone. In vivo, after gavage feeding rats, 7KC rapidly appeared in the liver and was converted to 7{beta}-hydroxycholesterol. CBX significantly decreased the ratio of 7{beta}-hydroxycholesterol to 7KC, supporting the evidence from cell culture experiments for 11{beta}-HSD1-dependent reduction of 7KC to 7{beta}-hydroxycholesterol. Upon inhibition of 11{beta}-HSD1 by CBX, 7KC tended to accumulate in the liver, and plasma 7KC concentration increased. Together, our results suggest that 11{beta}-HSD1 efficiently catalyzes the first step in the rapid hepatic metabolism of dietary 7KC, which may explain why dietary 7KC has little or no effect on the development of atherosclerosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Several in vitro studies demonstrated disturbances of cholesterol metabolism by 7KC1 (1), including effects on hydroxymethylglutaryl-CoA-reductase and cholesterol 7{alpha}-hydroxylase activities (2), inhibition of cholesterol release from cells (3), and down-regulation of low density lipoprotein receptor expression (4). In addition, 7KC exerts multiple effects by inhibiting nitric oxide release, decreasing glucose permeability, disrupting Ca2+-flux and inducing apoptosis in vascular cells (1).

Oxidized cholesterol metabolites play a potential role in the development of atherosclerosis (1, 5, 6). Among other oxysterols, 7KC is found at micromolar concentrations in human macrophage-foam cells and atherosclerotic lesions. In contrast, plasma 7KC concentrations are in the nanomolar range. The major oxysterols present in atherosclerotic plaques are 27-hydroxycholesterol and 7KC, although the direct role of 7KC in the development and progression of atherosclerosis is still unclear. 27-Hydroxycholesterol is produced by sterol 27-hydroxylase, the first enzyme of the alternative pathway from cholesterol to bile acids in the liver (7). 7KC is believed to be formed non-enzymatically by free radical oxidation of cholesterol (8) or absorbed from dietary intake of processed cholesterol-rich food (9, 10).

It was reported that upon administration, 7KC was rapidly metabolized in the liver and only little 7KC reappeared in the circulation, thus questioning the contribution of diet to the high oxysterol concentrations in atherosclerotic lesions (11, 12). Recent evidence suggested that sterol 27-hydroxylase is involved in 7KC metabolism. Patients with cerebrotendinous xanthomatosis have genetic defects in sterol 27-hydroxylase and develop atherosclerosis prematurely (13). They have normal circulating cholesterol levels but increased 7KC. Human monocyte-derived macrophages from healthy individuals but not from patients with cerebrotendinous xanthomatosis converted 7KC to 27-hydroxy-7KC (14). These results indicate an essential role for sterol 27-hydroxylase in the metabolism of 7KC in macrophage-foam cells. However, a recent study in sterol 27-hydroxylase-deficient mice demonstrated efficient hepatic metabolism of 7KC (15), thus indicating that another enzyme must be involved in the initial step of 7KC metabolism in the liver.

In addition to the formation of 7KC by auto-oxidation or its uptake from food, recent evidence from experiments with purified protein and liver microsomal fractions suggest that 7KC may be formed enzymatically from either 7{beta}-hydroxycholesterol or 7{alpha}-hydroxycholesterol in various species (16, 17). Song et al. (16) purified an enzyme from hamster liver microsomes, which efficiently converted both 7{alpha}-hydroxycholesterol and 7{beta}-hydroxycholesterol to 7KC. This enzyme also catalyzed the oxidation of corticosterone to 11-dehydrocorticosterone, and N-terminal sequencing of the purified enzyme revealed high similarity to human and rat 11{beta}-HSD1. However, unlike 11{beta}-HSD1, the purified hamster liver enzyme did not catalyze the reduction of 11-dehydrocorticosterone to corticosterone, and immunochemical analysis with an antibody raised against the purified enzyme suggested the existence of two distinct 11{beta}-HSD-like proteins in hamster liver. Moreover, Robinzon et al. (18) recently observed corticosterone inhibitable interconversion of the dehydroepiandrosterone (DHEA) metabolites 7{alpha}-DHEA, 7{beta}-DHEA, and 7-keto-DHEA in liver microsomal fractions, further suggesting a potential role of 11{beta}-HSD enzymes in the metabolism of 7-oxysterol compounds.

The role of 11{beta}-HSD1 in the local activation of glucocorticoid receptor by catalyzing the reduction of biologically inactive 11-ketoglucocorticoids (cortisone in humans, 11-dehydrocorticosterone in rodents) to active 11{beta}-hydroxyglucocorticoids (cortisol in humans, corticosterone in rodents) is well characterized (19, 20). Enhanced glucocorticoid levels lead to increased gluconeogenesis and antagonize the metabolic actions of insulin. Transgenic mice overexpressing 11{beta}-HSD1 selectively in adipose tissue have elevated adipose corticosterone concentrations, and they are susceptible to insulin-resistant diabetes, hyperlipidemia, and high arterial blood pressure due to an increased sensitivity to dietary salt and increased plasma levels of angiotensinogen, angiotensin II, and aldosterone (21, 22). Because adipose 11{beta}-HSD1 activity positively correlates to body mass index, percentage of body fat, and waist circumference as well as fasting glucose, insulin levels, and insulin resistance (23–25), 11{beta}-HSD1 is currently considered a promising pharmaceutical target for the treatment of diabetes type 2. Indeed, recent animal studies showed that the administration of a selective 11{beta}-HSD1 inhibitor to diabetic mice reduced blood glucose levels and increased insulin sensitivity (26, 27). In addition to its role in the activation of glucocorticoids, 11{beta}-HSD1 might play a role in the detoxification of reactive ketocompounds such as the potent tobacco carcinogen nicotine-derived nitrosamine ketone (NNK) and the anti-cancer drug oracin (28, 29).

In the present study we tested the hypothesis of whether 11{beta}-HSD1 plays a role in the interconversion of 7-hydroxycholesterol and 7KC. We compared recombinant rat, human and hamster 11{beta}-HSD1 in lysates and intact HEK-293 cells and measured the activities in rat and hamster liver homogenates and in intact rat hepatocytes and hamster liver tissue slices. We also studied the effect of the 11{beta}-HSD inhibitor CBX on 7KC metabolism in vivo in rats.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Cell culture media were purchased from Invitrogen, corticosterone, 11-dehydrocorticosterone, 7{alpha}-hydroxycholesterol, 7{beta}-hydroxycholesterol, and 7KC were from Steraloids (Wilton, NH), and [1,2,6,7-3H]corticosterone was from Amersham Biosciences. 11-[1,2,6,7-3H]Dehydrocorticosterone was prepared from radiolabeled corticosterone as described earlier (30). 27-Hydroxycholesterol was purchased from Medical Isotopes (Pelham, NH), and [1,2,6-3H]KC was from American Radiolabeled Chemicals, St. Louis, MO. Reagents for derivatization were obtained from Pierce. All other chemicals were from Fluka AG (Buchs, Switzerland) and were of the highest grade available.

Cloning of Hamster 11{beta}-HSD1—The liver from a Golden Syrian Hamster (Charles River Laboratories) was snap-frozen in liquid nitrogen followed by extraction of total RNA from 100 mg of tissue using Trizol reagent according to the instructions by the manufacturer (Invitrogen). Hamster 11{beta}-HSD1 was cloned by reverse transcription of 5 µg of total RNA with primer XRT20 (5'-TATTCTAGACACCTGAGCACGT20-3') and subsequent PCR amplification using Taq polymerase:Pfu polymerase (10:1), primer XR (5'-TATTCTAGACACCTGAGCACG-3'), and a degenerate primer (5'-AAAGGATCCGCCTCCCTGTCTGATG(C/G)(A/C)(C/T)TT(T/C)ATG-3') based on comparison with the sequences upstream of the initiation codon of 11{beta}-HSD1 from other species. A single DNA product was obtained, cleaved with BamHI and XbaI, and cloned into Bluescript vector. The sequences of all 10 clones analyzed were identical. The cDNA insert with or without a C-terminal FLAG epitope tag that was attached by PCR, was inserted into the pcDNA3 expression vector.

Preparation of Liver Homogenates—The livers from male Sprague-Dawley rats or, alternatively, from Golden Syrian hamsters, were removed, immersed in phosphate-buffered saline at 4 °C, cut in small pieces, and homogenized in buffer TG1 (20 mM Tris-HCl, pH 7.4, 1 mM EGTA, 1 mM EDTA, 1 mM MgCl2, 100 mM NaCl, 20% glycerol) (5 ml/g) using a Potter-Elvehjem homogenizer. The homogenates were subjected to a single centrifugation step at 10,000 x g for 10 min at 4 °C. The supernatants were adjusted to a protein concentration of 0.1 mg/ml, and activities were determined immediately.

Isolation of Rat Hepatocytes—Male Sprague-Dawley rats (200–250 g body weight) were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital. Heparin (liquemin), 1 ml/kg, was injected into the vena cava followed by perfusion for 15 min with 16 ml/min of prewarmed, oxycarbon-aerated buffer containing 10 mM HEPES, pH 7.4, 143 mM NaCl, 7 mM KCl, and 0.2 mM EGTA. The liver was then perfused for 20 min with 10 ml/min of a buffer containing 50 mM HEPES, pH 7.6, 100 mM NaCl, 7 mM KCl, 5 mM CaCl2, and 0.1% (w/v) collagenase (Clostridium histolyticum type IV, Sigma). The liver was removed and incubated in a buffer containing 10 mM HEPES, pH 7.4, 0.8 mM Na2HPO4, 135 mM NaCl, 5 mM KCl, 0.8 mM MgSO4, 1.2 mM CaSO4, and 5 mM glucose, the capsule was opened, and hepatocytes were harvested. Cells were incubated with slight shaking for 10 min at 37 °C. Hepatocytes were filtered through a 100-µm pore size nylon mesh to separate from biliocytes, washed twice in the same buffer, and centrifuged for 2 min at 50 x g. Cells were finally suspended in steroid-free Dulbecco's modified Eagle's medium, and enzymatic activities were measured immediately.

Preparation of Intact Hamster Liver Slices—Hamster livers were removed, rinsed thoroughly with Hanks' buffered salt solution, and cut in thin slices of ~300 µm with parallel razor blades similar to the method for precision-cut liver slices described by Dogterom and Rothuizen (31). Slices were rinsed with Hanks' buffer, and activities were measured immediately without adding NADPH. When the slices were lysed, no conversion of 7KC was detected unless NADPH was added to the reaction.

Determination of Enzyme Activities—HEK-293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum followed by transient transfection with rat 11{beta}-HSD1 expression plasmid (32). Cells were washed three times 24 h post-transfection with steroid-free (double charcoal-treated) medium and grown for another 24 h. Cells were then detached with phosphate-buffered saline, centrifuged for 3 min at 150 x g, and resuspended in the appropriate volume of TG1 buffer. Cells were lysed by sonication, and activities were determined immediately.

11{beta}-HSD1 activities were measured as described recently, with minor modifications (30). Briefly, lysates were incubated in TG1 buffer containing 500 µM NADP+ or NADPH, 30 nCi of [1,2,6,7-3H]corticosterone or 11-[1,2,6,7-3H]dehydrocorticosterone, and various concentrations of unlabeled steroids ranging from 20 nM to 2 µM. Samples in a final volume of 20 µl were incubated at 37 °C for 10–30 min, reactions were stopped, and steroids were separated by thin-layer chromatography (TLC) and analyzed by scintillation counting. When measuring activities in intact cells, steroid-free medium was used and cofactor was omitted.

7-Hydroxydehydrogenase and 7-oxoreductase activities in cell lysates or liver homogenates were measured in buffer TG1 in the presence of various concentrations of 7{alpha}-hydroxycholesterol, 7{beta}-hydroxycholesterol, or 7KC ranging from 50 nM to 2.5 µM and the corresponding cofactor in a final reaction volume of 1 ml. Reactions were incubated for 10–30 min at 37 °C and stopped by adding 7 ml of dichloromethane. Intact transfected HEK-293 cells or rat hepatocytes were incubated in 1 ml of steroid-free medium containing the corresponding cholesterol metabolite and stopped as mentioned above. Samples were then subjected to derivatization and analysis by gas chromatography-mass spectrometry (GC-MS). Alternatively, the reduction of 7KC in intact rat hepatocytes or in intact hamster liver slices was measured by adding 50 nCi of radiolabeled 7KC as a tracer followed by determination of the conversion after separation of 7-oxycholesterols by TLC.

Thin-layer Chromatography—Radiolabeled corticosterone and 11-dehydrocorticosterone were separated by TLC (SIL G-25 UV254, Macherey-Nagel, Oensingen, Switzerland) using a solvent system of 9:1 (v/v) chloroform:methanol. A solvent system 2:3 (v/v) hexane:ethyl acetate was used to separate radiolabeled 7KC, 7{alpha}-hydroxycholesterol, and 7{beta}-hydroxycholesterol. 7KC was detected under UV light, whereas 7{alpha}-hydroxycholesterol and 7{beta}-hydroxycholesterol were visualized using a molybdate spray (100 ml of 10% H2SO4,5gof(NH4)Mo7O24·6H2O, 0.1 g of Ce(SO4)2).

Animal Experimentation—Written approval for the present experiments was obtained from the Ethical Committee for Animal Research of the University of Berne, Switzerland. Male Sprague-Dawley rats (200–250 g body weight) obtained from Charles River Laboratories, were fed standard laboratory chow ad libitum. Groups of five animals each received 2 mg/kg of body weight of 7KC by gavage feeding. Animals were sacrificed after 1, 2, 4, 6, or 8 h, and plasma and tissues were harvested. To study the effect of the 11{beta}-HSD inhibitor CBX, animals were intraperitoneally injected with 15 mg/kg of body weight of CBX 1 h before administration of 7KC and 2 and 5 h after 7KC administration, respectively. For analysis of plasma electrolytes and steroid hormones, groups of four animals were treated as above and sacrificed 6 h after 7KC administration. Plasma aldosterone and progesterone were measured by radioimmunoassay using the coat-a-count procedure as described by the manufacturer (Diagnostic Products Corp., Los Angeles, CA). Other steroid metabolites were determined by GC-MS as described previously (33).

Analysis of 7-Oxycholesterols by GC-MS—After removal, tissues were washed from contaminating blood with phosphate-buffered saline, blotted on filter paper, snap-frozen in liquid nitrogen, and stored at –80 °C until analysis. 7-Oxycholesterols were analyzed according to the method described by Iuliano et al. (34) with modifications. Tissues (100–250 mg) were cut into small pieces and homogenized in a solution containing 10 ml of chloroform:methanol (2:1, v/v), 0.01% butylated hydroxytoluene (v/w) and transferred to a tube containing 2 ml of 0.9% NaCl (H2O), methanol (19:1) and 100 ng of 6{alpha}-hydroxycholestanol (5{alpha}-cholestan-3{beta},6{alpha}-diol) as the internal standard. Plasma samples (1 ml), obtained from EDTA anti-coagulated blood, were treated as above but without homogenization. After mixing, samples were centrifuged at 750 x g for 5 min at 4 °C. The organic phase was washed twice with 4 ml of methanol, 0.9% NaCl (1:1, v/v) and centrifuged, and the organic phase was subjected to alkaline hydrolysis (saponification) with 0.35 M KOH under an argon atmosphere for 2 h at 25 °C. The reaction mixture was adjusted to pH 7.0 with phosphoric acid and centrifuged, and the organic phase was washed twice with 2 ml of 0.9% NaCl (H2O), methanol (19:1). The organic phase was transferred to a new tube containing 100 µl of 1% butylated hydroxytoluene in pyridine. For the external standard 125 ng of stigmasterol was added followed by evaporation of the solvent. Next, samples were loaded on a silica cartridge (Supelco), and cholesterol was removed with 0.5% 2-propanol in cyclohexane. Oxidized cholesterol metabolites were eluted with 30% 2-propanol in cyclohexane into a tube containing 100 µl of 1% butylated hydroxytoluene in pyridine, the solvent was evaporated under nitrogen, and the sample was converted to trimethylsilyl ether by adding 100 µl of pyridine and 100 µl of N,O-bis(trimethylsilyl)trifluoroacetamide. Samples were incubated under argon for 1 h at 60 °C. 200 µl of cyclohexane were added to the residue, and the sample was subjected to GC-MS analysis.

Enzyme activities from cell lysates or intact cells were measured in 1-ml reaction volumes and stopped by the addition of 7 ml of dichloromethane. The internal standard medroxyprogesterone (0.5 µg) was added, and samples were mixed for 20 min at 4 °C followed by centrifugation for 5 min at 2000 x g. The organic phase was transferred to new tubes, and dichloromethane was evaporated at 25 °C under argon. For the external standard 0.5 µg of stigmasterol was added. The solvent was evaporated, and 100 µl of 2% methoxamine-HCl in pyridine was added to protect keto groups followed by incubation for 1 h at 60 °C. After evaporation of the solvent, 100 µl of trimethylsilylimidazole was added to protect hydroxyl groups followed by incubation for 16 h at 100 °C. After cooling to 25 °C, 500 µl of cyclohexane:pyridine:hexamethyldisilazane (98:1:1) was added, and samples were purified on Lipidex-5000 columns. Another 2.5 ml of the above mixture were added, samples were collected, and the solvent was evaporated. Cholesterol metabolites were dissolved in 200 µl of cyclohexane, sonicated for 1 min, and subjected to GC-MS analysis on a Hewlett Packard gas chromatograph 6890 equipped with a mass-selective detector 5973 by selected ion monitoring).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of Hamster Liver 11{beta}-HSD1—To test whether 11{beta}-HSD1 accepts both glucocorticoids and 7-oxycholesterols as substrates and whether the hamster 7{alpha}-hydroxycholesterol dehydrogenase purified by Song et al. (16) is indeed 11{beta}-HSD1, we compared the activities of rat, human, and hamster 11{beta}-HSD1. The coding region of 11{beta}-HSD1 was cloned from liver RNA of a Golden Syrian hamster. PCR amplification yielded a single fragment (accession number AY519498 [GenBank] ) encoding a protein of 292 amino acids. Fig. 1 shows ~90% identity between hamster, rat, and human 11{beta}-HSD1. The N-terminal sequence of hamster 11{beta}-HSD1 is identical to the peptide sequence of the 7{alpha}-hydroxycholesterol dehydrogenase purified by Song et al. (16), demonstrating that their purified protein is indeed 11{beta}-HSD1 (16).



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  		FIG. 1.
Alignment of the protein sequences of hamster, human, and rat 11{beta}-HSD1 (accession numbers: AY519498 [GenBank] , NM_ 005525, and NM_017080 [GenBank] ). Identical residues are shaded. The conserved tyrosine and lysine residues in the catalytic center (*), the transmembrane helix (—), and the conserved glycine residues of the Rossmann-fold in the cofactor binding region (+) are indicated.

 
Comparison of Recombinant Rat, Human, and Hamster 11{beta}-HSD1 Activities—Recombinant rat, human, and hamster 11{beta}-HSD1 were expressed in HEK-293 cells, and activities in cell lysates were determined. Untransfected cells neither interconverted glucocorticoids nor 7-oxycholesterols. 11{beta}-HSD1 from all three species efficiently catalyzed both the oxidation of corticosterone and the reduction of 11-dehydrocorticosterone (Table I). The activities in the three species were comparable, except that human 11{beta}-HSD1 catalyzed the oxidation of corticosterone with ~3-fold lower apparent Km and 3-fold lower apparent Vmax. This was also observed when activities were measured in intact cells (Table II). The reduction of 11-dehydrocorticosterone in intact cells expressing recombinant 11{beta}-HSD1 was comparable in all three species.


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  		TABLE I
Oxidation and reduction by lysates from HEK-293 cells expressing recombinant 11{beta}-HSD1

Enzymatic activities of lysates from HEK-293 cells transiently transfected with either human, rat or hamster 11{beta}-HSD1 were determined by measuring the reduction of 11-dehydrocorticosterone and 7KC in the presence of NADPH or the oxidation of corticosterone and 7-hydroxycholesterols in the presence of NADP+ as described under "Experimental Procedures." Data represent the mean ± S.D. and were obtained from at least four independent experiments. ND, measurements were below the detection limit. NA, not analyzed.

 


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  		TABLE II
Oxidation and reduction of glucocorticoids and 7-oxycholesterols in intact HEK-293 cells expressing recombinant 11{beta}-HSD1

Enzymatic activities in intact HEK-293 cells transiently expressing either human, rat, or hamster 11{beta}-HSD1 were determined by measuring the reduction of 11-dehydrocorticosterone and 7KC or the oxidation of corticosterone and 7-hydroxycholesterols in steroid-free medium as described under "Experimental Procedures." Data represent the mean ± S.D. and were obtained from at least four independent experiments.

 
Lysates expressing recombinant rat or human 11{beta}-HSD1 stereospecifically oxidized 7{beta}-hydroxycholesterol to 7KC (Table I). The hamster enzyme efficiently oxidized both 7{alpha}-hydroxycholesterol and 7{beta}-hydroxycholesterol to 7KC, with a slight preference for 7{alpha}-hydroxycholesterol. All three species efficiently catalyzed the reduction of 7KC, whereby rat and human 11{beta}-HSD1 acted stereo-specifically, forming exclusively 7{beta}-hydroxycholesterol, whereas hamster 11{beta}-HSD1 led to the formation of both 7{alpha}-hydroxycholesterol (60%) and 7{beta}-hydroxycholesterol (40%). Untransfected HEK-293 cells or cells transfected with rat or human 11{beta}-HSD2 were unable to reduce 7KC.

Intact cells expressing rat, human, or hamster 11{beta}-HSD1 efficiently catalyzed the reduction of 7KC; however, in contrast to lysates, 11{beta}-HSD1 did not act as 7-hydroxycholesterol dehydrogenase in intact cells (Table II). The rat and human enzymes were stereo-specific, whereas hamster 11{beta}-HSD1 led to the formation of 7{alpha}-hydroxycholesterol and 7{beta}-hydroxycholesterol (60 and 40%, respectively). As observed with lysates, apparent Km values were similar, whereas Vmax of the reduction of 7KC was about 30% that of the reduction of 11-dehydrocorticosterone. A graphic plot of the activities from rat 11{beta}-HSD1 suggests that the enzyme follows first order rate kinetics (Fig. 2).



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  		FIG. 2.
Metabolism of 7-oxycholesterols by recombinant rat 11{beta}-HSD1. Enzymatic activities of recombinant rat 11{beta}-HSD1 were determined in lysates or in intact HEK-293 cells as described under "Experimental Procedures." The insets show Lineweaver-Burk plots of the concentration-dependent oxidation of 7{beta}-hydroxycholesterol in lysates (A) or reduction of 7KC in lysates (B) or in intact cells (C). Representative experiments are shown.

 
Comparison of Endogenous Rat and Hamster Liver 11{beta}-HSD1 Activities—We then compared 11{beta}-HSD1-dependent activities under endogenous conditions using rat and hamster liver homogenates. The results presented in Table III confirmed those obtained with lysates from HEK-293 cells expressing recombinant 11{beta}-HSD1 (Table I), except that Vmax values were higher. The oxidative activities remained relatively stable during homogenization and subsequent centrifugation. In contrast, a loss of reductase activities was observed, which was more pronounced for 7KC compared with 11-dehydrocorticosterone. A single centrifugation step was used after homogenization of the liver, and activities were determined immediately because a time-dependent loss of the reductase activities was observed. Isolated microsomes retained oxidative activities and some reductase activity for 11-dehydrocorticosterone, whereas reduction of 7KC was completely abolished under the conditions applied (not shown).


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  		TABLE III
Metabolism of glucocorticoids and 7-oxycholesterols by rat and hamster liver homogenates

Enzymatic activities were determined by measuring the reduction of 11-dehydrocorticosterone and 7KC in the presence of NADPH or the oxidation of corticosterone and 7-hydroxycholesterols in the presence of NADP+ as described under "Experimental Procedures." Data represent the mean ± S.D. and were obtained from at least four independent experiments.

 
Next, we measured activities in isolated intact rat primary hepatocytes and in intact hamster liver tissue slices. In both systems no oxidative activities could be detected with 7{beta}-hydroxycholesterol or 7{alpha}-hydroxycholesterol as substrates even after prolonged incubation and at various concentrations. Intact hepatocytes catalyzed the stereospecific reduction of 7KC to 7{beta}-hydroxycholesterol (Fig. 3, A and B), and intact hamster liver slices converted 7KC to 7{alpha}-hydroxycholesterol and 7{beta}-hydroxycholesterol (62 and 38% after 30 min) (Fig. 3, C and D), comparable with the results obtained with recombinant 11{beta}-HSD1 in the HEK-293 expression system (Table II). Fig. 3 shows that the reduction of 7KC in intact rat hepatocytes and hamster liver slices was almost completely blocked by adding 10 µM nonselective 11{beta}-HSD inhibitor CBX or by 200 µM concentrations of the selective 11{beta}-HSD1 inhibitor 2'-hydroxyflavanone (30). These results together with the findings from experiments with transfected HEK-293 cells indicate that the reduction of 7KC to 7-hydroxycholesterol in the liver is predominantly catalyzed by 11{beta}-HSD1.



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  		FIG. 3.
Metabolism of 7KC in intact rat hepatocytes and in intact hamster liver tissue slices. Freshly isolated rat hepatocytes (A and B) or hamster liver slices (C and D) were preincubated for 15 min with 10 µM CBX or 200 µM 2'-hydroxyflavanone (2-Flav) as indicated followed by incubation in steroid-free medium with 800 nM 7KC for 30 min (A and C) or 90 min (B and D) at 37 °C. 7-Oxycholesterol metabolites were analyzed by GC-MS as described under "Experimental Procedures." White bars, 7KC; black bars, 7{beta}-hydroxycholesterol; hatched bars, 7{alpha}-hydroxycholesterol. Results are expressed as a percentage of initially supplied 7KC.

 
Comparison of the amounts of 7KC and 7-hydroxycholesterol after incubation for 30 min (Fig. 3, A and C) and 90 min (Fig. 3, B and D) shows that 7KC disappeared more rapidly than 7-hydroxycholesterol content increased, suggesting that intact liver cells further metabolized 7-hydroxycholesterol. In addition, ~25% of 7KC was metabolized to substrates other than 7{beta}-hydroxycholesterol after 90 min in the presence of either CBX or 2'-hydroxyflavanone. Therefore, 7KC seems to be mainly metabolized by 11{beta}-HSD1, but other enzymes such as sterol 27-hydroxylase are likely to be involved in the metabolism of 7KC.

11{beta}-HSD1-dependent Reduction of 7KC to 7{beta}-Hydroxycholesterol in Vivo in Rats—To study the role of 11{beta}-HSD1 in the metabolism of 7KC in vivo, we administered 7KC (2 mg/kg body weight) by gavage feeding to male Sprague-Dawley rats and determined the amount of 7-oxycholesterols in plasma and liver. 7KC rapidly appeared in the liver, reaching the highest concentrations of about 850 ng/g of tissue 6 h after administration (Fig. 4A). In livers from rats treated with 15 mg/kg of body weight CBX, 7KC concentrations tended to increase more rapidly than in untreated animals and reached ~2-fold higher concentrations after 6 and 8 h. The observed tendency of CBX-dependent accumulation of 7KC was paralleled by a reduced formation of 7{beta}-hydroxycholesterol in CBX-treated rats with maximal levels of 700 ng/g of tissue after 6 h (Fig. 4B). In contrast, in the absence of CBX, 7{beta}-hydroxycholesterol concentrations increased rapidly to a maximal level of 1550 ng/g of tissue after 6 h followed by a decline to 1000 ng/g of tissue after 8 h. Despite large interindividual differences, the values of 7{beta}-hydroxycholesterol in the absence of CBX were significantly higher than the corresponding values in the presence of the inhibitor at 2, 4, and 6 h after the administration of 7KC (p < 0.05).



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  		FIG. 4.
Rapid metabolism of 7KC in the liver. 7KC (2 mg/kg body weight) was administered by gavage feeding to either untreated male Sprague-Dawley rats (200–220 g) ({blacktriangleup}) or to animals treated 1 h before 7KC administration and after 2 h and 5 h with 15 mg/kg of the 11{beta}-HSD inhibitor CBX ({square}). Animals were sacrificed at time 0 or after 1, 2, 4, 6, or 8 h, the livers were removed, and whole liver homogenates were analyzed for the content of 7-oxycholesterols as described under "Experimental Procedures." 7KC concentrations (A) and 7{beta}-hydroxycholesterol concentrations (B) in livers from untreated ({blacktriangleup}) or CBX-treated rats ({square}) are shown. C, ratio of 7{beta}-hydroxycholesterol to 7KC in livers from untreated ({blacktriangleup}) and CBX treated rats ({square}). D, plot of 7KC against 7{beta}-hydroxycholesterol content from livers of untreated ({blacktriangleup}) and CBX-treated rats ({square}). Values are expressed as mean ± S.D. (4 or 5 rats/time point).

 
The comparison of the concentrations of 7KC and 7{beta}-hydroxycholesterol in the absence or presence of CBX revealed a positive correlation of 7KC to 7{beta}-hydroxycholesterol in the in vivo experiment with a highly significant shift from 7{beta}-hydroxycholesterol to 7KC in the presence of the 11{beta}-HSD inhibitor CBX (7{beta}-hydroxycholesterol:7KC, absence versus presence of CBX; p < 10E–10)(Fig. 4, C and D). The ratio of 7{beta}-hydroxycholesterol to 7KC was significantly higher in the absence of CBX than in its presence (p < 0.05) at all time points except at time 0.

Upon administration of 7KC, plasma concentrations of 7{beta}-hydroxycholesterol increased to 200 ng/ml after 2 h, stayed more or less constant for another 4 h, and then declined (Fig. 5A). CBX caused an increase in plasma 7KC concentrations with maximal levels of 330 ng/ml after 6 h. The increase in 7KC concentration was significant (p < 0.05) after 8 h. CBX treatment also reduced the occurrence of 7{beta}-hydroxycholesterol in plasma, although these differences were not significantly different (Fig. 5B). The ratio of 7{beta}-hydroxycholesterol to 7KC was significantly lower after 4, 6, and 8 h in animals cotreated with CBX compared with animals treated only with 7KC (Fig. 5, C and D). These results suggest reduced removal of 7KC from plasma, most likely due to reduced metabolism in tissues expressing 11{beta}-HSD1.



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  		FIG. 5.
Determination of 7KC and 7{beta}-hydroxycholesterol in plasma. Rats were treated as described in Fig. 3, and the content of 7-oxycholesterols were determined in plasma (see "Experimental Procedures"). Shown are 7KC concentrations (A) and 7{beta}-hydroxycholesterol concentrations (B) in plasma from untreated ({blacktriangleup}) or CBX-treated rats ({square}). C, ratio of 7{beta}-hydroxycholesterol to 7KC in plasma from untreated ({blacktriangleup}) and CBX-treated rats ({square}). D, plot of 7KC against 7{beta}-hydroxycholesterol content from plasma of untreated ({blacktriangleup}) and CBX-treated rats ({square}). Values are expressed as mean ± S.D. (4 or 5 rats per time point).

 
Reduction of 7KC to 7{beta}-Hydroxycholesterol by 11{beta}-HSD1 in Homogenates from Major Organs in the Rat—To assess the relative contribution of 11{beta}-HSD1-dependent reduction of 7KC to 7{beta}-hydroxycholesterol by the major organs in the rat, we determined the reduction of 7KC in homogenates from liver, kidney, heart, and intestine (duodenum) in the absence or presence of the nonselective 11{beta}-HSD inhibitor CBX and the selective 11{beta}-HSD1 inhibitor 2'-hydroxyflavanone. Upon incubation of homogenates for 30 min at 37 °C with 800 nM 7KC, activities of 1.8 and 0.4 nmol x h–1 x mg–1 were obtained for liver and kidney, respectively, whereby CBX (10 µM) and 2'-hydroxyflavanone (200 µM) inhibited more than 90% of the generation of 7{beta}-hydroxycholesterol. Activities for heart and intestine (duodenum) were below 0.05 nmol x h–1 x mg–1.

CBX Does Not Disturb Plasma Electrolytes and Steroid Hormone Levels under the Conditions Used—In the in vivo experiment the effect of CBX on 7{beta}-hydroxycholesterol generation was most pronounced 6 h after 7KC administration. To investigate whether the doses of the nonselective 11{beta}-HSD inhibitor CBX applied in the present study significantly increased corticosterone concentrations by inhibition of 11{beta}-HSD2 or interfered with other steroid hormone-metabolizing enzymes, we compared electrolytes, creatinine, and various steroid hormones in the plasma of control rats, animals treated for 6 h with 7KC, CBX, or both compounds. No significant changes were observed in these essential parameters within the time course of the experiment (Table IV). Importantly, the ratio of plasma corticosterone to 11-dehydrocorticosterone, a measure for the combined activities of 11{beta}-HSD1 and 11{beta}-HSD2, was not altered, indicating little or no inhibition of 11{beta}-HSD2 within the relatively short time of treatment.


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  		TABLE IV
Plasma concentrations of electrolytes, creatinine, and steroid hormones after CBX treatment

Sprague-Dawley rats received 2 mg/kg 7KC by gavage feeding with or without treatment with 15 mg/kg CBX every 3 h. Six hours after the administration of 7KC animals were sacrificed, plasma was obtained, and concentrations of electrolytes, creatinine, and steroid hormones were determined as described under "Experimental Procedures." Data represent the mean ± S.D. (n = 4).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Despite the pro-atherogenic effects of oxysterols and their presence in micromolar concentrations in human macrophage-foam cells and atherosclerotic lesions (1, 5), there is evidence emerging that dietary oxysterols play a rather minor role in atherogenesis due to their rapid hepatic metabolism (11, 12, 35). Sterol 27-hydroxylase was shown to hydroxylate 7KC to 27-hydroxy-7KC in human atherosclerotic lesions and in cultured macrophages (14); however, only a transient increase in aortic levels of 7KC was observed in sterol 27-hydroxylase-deficient mice after 7KC administration, which was followed by efficient hepatic metabolism (15). These results suggested the existence of an as yet unidentified mechanism contributing to the clearance of 7KC in the liver.

Here, we describe a novel potential mechanism for the first step in the hepatic metabolism of 7KC after its uptake from food. Together, our results from in vitro studies with recombinant 11{beta}-HSD1, endogenous 11{beta}-HSD1 in liver homogenates, or in intact liver cells as well as from in vivo experiments with rats demonstrate an important role of 11{beta}-HSD1 in the detoxification of food-derived 7KC in the liver. Although lysates from cells expressing recombinant 11{beta}-HSD1 catalyzed both oxidation of 7-hydroxycholesterol and reduction of 7KC, the enzyme exclusively catalyzed the reduction reaction in intact cells. The stereo-specific reduction of 7KC to 7{beta}-hydroxycholesterol by rat and human 11{beta}-HSD1 and to 7{alpha}-hydroxycholesterol and 7{beta}-hydroxycholesterol by hamster 11{beta}-HSD1 was comparable in lysates and intact cells expressing recombinant enzymes as well as in homogenates and intact hepatocytes or liver tissue slices expressing endogenous 11{beta}-HSD1. CBX, a nonselective inhibitor of 11{beta}-HSD enzymes, and 2'-hydroxyflavanone, a selective 11{beta}-HSD1 inhibitor (30), both reduced enzymatic activities in HEK-293 cells expressing recombinant 11{beta}-HSD1 and in cells expressing endogenous 11{beta}-HSD1. These results provide strong evidence that 11{beta}-HSD1 acts as a 7KC reductase but not as a 7-hydroxycholesterol dehydrogenase under endogenous conditions. The absolute kinetic parameters obtained from experiments with cultured cells, however, have to be analyzed with caution, since the addition of the free compound to the culture medium may not reflect the physiological situation.

The cloning of hamster 11{beta}-HSD1 and its comparison with the rat and human enzyme explains part of the species-specific differences in the interconversion of 7-hydroxycholesterol and 7KC observed by Maeda et al. (17). They observed the oxidation of 7{alpha}-hydroxycholesterol to 7KC by hamster and chicken but not rat, rabbit, or guinea pig liver microsomes and conversion of 7{beta}-hydroxycholesterol to 7KC in all species except guinea pig. Importantly, Maeda et al. (17) report the reduction of 7KC to both 7{alpha}-hydroxycholesterol and 7{beta}-hydroxycholesterol in equal amounts by hamster liver microsomes, consistent with the activities of recombinant hamster 11{beta}-HSD1 obtained in the present study. This is, however, in contrast to Song et al. (16), reporting only oxidation of 7-hydroxycholesterols. Because the hamster enzyme purified by Song et al. (16) did not reduce 11-dehydrocorticosterone, it is possible that reductase activity was lost during microsomal preparation or purification, in line with the present observation of a loss of reductase activity upon preparation of microsomes.

Recent analyses by Prough and coworkers (18, 36) showing the inhibition of the interconversion of 7-oxy-DHEA metabolites by corticosterone suggested the involvement of 11{beta}-HSD enzymes. These studies described both rat and human liver microsomes as converting 7{alpha}-hydroxy-DHEA to 7-keto-DHEA, whereby the human enzyme was ~7-fold more efficient. The reverse reaction was also catalyzed; however, at much lower efficiency. In addition, Prough and coworkers (18, 36) report conversion of 7{alpha}-hydroxy-DHEA to 7-keto-DHEA by kidney microsomes and suggest that this reaction was catalyzed by 11{beta}-HSD2 or by an as yet unidentified 11{beta}-HSD3. In the present study, we demonstrate that human and rat 11{beta}-HSD1 catalyze the stereospecific interconversion of 7{beta}-hydroxycholesterol and 7KC, whereby only the reduction of 7KC is catalyzed in intact cells. These experiments indicate significant differences between 7KC and 7-keto-DHEA metabolism regarding the isozyme involved and the stereospecificity as well as the tissue specificity of the corresponding reaction. Clearly, experiments with recombinant enzymes and the use of intact cells are required to elucidate species-specific differences between 7-oxy-DHEA metabolites and 7-oxycholesterols, to reveal the isozyme responsible for the observed activity in the corresponding tissue, and to assess the reaction direction.

Oxidized cholesterol products including 7KC are present at high concentrations in processed cholesterol-rich food (9). A recent comparison of 7-oxycholesterol compounds in fresh versus broiled olive oil and butter revealed high 7KC concentrations of 30 µM and 7{alpha}-hydroxycholesterol and 7{beta}-hydroxycholesterol concentrations of 10 µM in broiled butter compared with concentrations clearly below 0.5 µM in fresh or broiled olive oil and in fresh butter.2 Upon ingestion, dietary oxysterols are absorbed through the intestine and incorporated into lymph chylomicrons (10, 37, 38) followed by partial hydrolysis of the triacylglycerol core and clearance of the chylomicron remnants by hepatocytes (39). Lyons and Brown (12) observe rapid hepatic metabolism of 7KC when delivered in either acetylated low density lipoprotein or in chylomicron remnant-like emulsion to rats.

Our results from the in vivo experiments with rats suggest that the initial step in the metabolism of 7KC, which is incorporated into chylomicrons and transported into the liver, is the stereo-specific conversion to 7{beta}-hydroxycholesterol catalyzed by 11{beta}-HSD1. The rapid hepatic conversion of 7KC to 7{beta}-hydroxycholesterol in rats was significantly reduced by CBX, although the concentration applied (15 mg/kg) did not lead to complete inhibition. CBX also inhibits 11{beta}-HSD2; however, it is not expressed in liver and does not reduce 7KC. Studies in vivo in man and in animals provided evidence that CBX predominantly inhibits 11{beta}-HSD1 in the liver but not in adipose tissue (40). Treatment of humans with CBX for 1 week decreased the ratio of urinary tetrahydrocortisol and allo-tetrahydrocortisone to tetrahydrocortisone, indicating inhibition of hepatic 11{beta}-HSD1, and slightly increased the ratio of urinary free cortisol to cortisone, indicating modest inhibition of 11{beta}-HSD2 (41). In the present study, inhibition of 11{beta}-HSD2 in the kidney was not relevant since plasma corticosteroid levels were not altered. In addition, CBX did not alter plasma concentrations of electrolytes or various other steroids, suggesting that the observed effect of CBX on 7KC metabolism is due to inhibition of 11{beta}-HSD1 and not caused by a secondary effect through another steroid-metabolizing enzyme. The interference of other effects of CBX, including inhibition of collagen-{alpha}1 expression (42), stimulation of intestinal bicarbonate secretion (43), and action as a gap-junction uncoupler (44) on 7KC metabolism, cannot be fully excluded; however, these effects were either observed after prolonged exposure or at high concentrations (>50 µM in cell experiments; >50 mg/kg in animal experiments).

A comparison of the conversion of 7KC to 7{beta}-hydroxycholesterol by major rat organs showed that the liver has by far the highest activity, followed by 4–5-fold lower activity in kidneys and barely detectable activities in heart and intestine. Because both CBX and 2'-hydroxyflavanone abolished the conversion of 7KC to 7{beta}-hydroxycholesterol in all tissue homogenates examined, these results suggest that this activity is mainly catalyzed by hepatic 11{beta}-HSD1. 2'-Hydroxyflavanone, a selective 11{beta}-HSD1 inhibitor without significant effects on 11{beta}-HSD2, 17{beta}-HSD1, or 17{beta}-HSD2, is not suitable for the inhibition of 11{beta}-HSD1 in vivo in rats, because it is a weak inhibitor of rat 11{beta}-HSD1 (IC50 of 80 µM) compared with human 11{beta}-HSD1 (IC50 of 1.8 µM).3

Our finding that 11{beta}-HSD1 stereo-specifically converts 7KC to 7{beta}-hydroxycholesterol in rats is supported by previous studies reporting the generation of 7{beta}-hydroxycholesterol after intraperitoneal or intravenous administration of 7KC to rats (45–47) and by a study from Lyons et al. (11) who observed rapid hepatic metabolism upon administration of radiolabeled 7KC. Their TLC analysis revealed the transient occurrence of 7{beta}-hydroxycholesterol but not 7{alpha}-hydroxycholesterol, indicating stereospecific conversion of 7KC to 7{beta}-hydroxycholesterol, further metabolism of the latter compound, formation of bile acids, and excretion.

The initial increase in 7KC content observed in the rat liver 6 h after administration (Fig. 4A) may reflect hepatic 7KC uptake from the clearance of chylomicrons after intestinal absorption, and the subsequent decline may be explained by hepatic conversion to 7{beta}-hydroxycholesterol, other metabolites including 27-hydroxy-7KC, and uptake by other tissues. Fig. 3 shows that CBX and 2'-hydroxyflavanone both abolished 7KC metabolism in intact rat hepatocytes and hamster liver tissue slices, with only about 25% of 7KC metabolized to other compounds after incubation for 90 min. The rapid decline of 7{beta}-hydroxycholesterol after 6 h likely reflects the rapid metabolism of this compound in the liver (Fig. 4B). In intact rat hepatocytes, the decrease in 7KC, and the increase in 7{beta}-hydroxycholesterol concentration was almost equal after 30 min, but after 90 min 7{beta}-hydroxycholesterol content was only about 50% of the amount expected (Fig. 3), indicating further metabolism of 7{beta}-hydroxycholesterol. Similarly, in intact hamster liver slices the decrease in 7KC was accompanied by an increase in 7{alpha}-hydroxycholesterol and 7{beta}-hydroxycholesterol, and both compounds seemed to undergo further metabolism.

Sterol 27-hydroxylase-deficient mice have typically decreased fecal bile acid metabolites; however, fecal excretion of 7KC metabolites was increased, indicating an unusual bile acid pathway. Norii et al. (48) found that upon intraperitoneal administration of 2 mg of radiolabeled 7{beta}-hydroxycholesterol to rats, most of the compound was metabolized and excreted in bile as acidic fraction, with the main metabolites being 3{alpha},7{beta}-dihydroxy-5-cholenoic acid and 3{beta},7{beta}-dihydroxy-5-cholenoic acid. These authors also reported the excretion of 3-hydroxy-7-oxo-5-cholenoic acid; however, their saponification conditions (130 °C for 3 h) suggest that the 7-oxo compounds were formed by auto-oxidation rather than enzymatically. Ursodeoxycholic acid, the 7{beta}-epimer of chenodeoxycholic acid, was found as a minor metabolite only, indicating that 7{beta}-hydroxycholesterol follows an unusual metabolic pathway. 3{beta},7{beta}-Dihydroxy-5-cholenoic acid has also been identified as a 7-N-acetylglucosamine conjugate and shown to be a minor component in the urine of healthy individuals (49, 50). These unusual bile acid metabolites may be derived from 7{beta}-hydroxycholesterol after its hepatic conversion from 7KC by 11{beta}-HSD1.

It is tempting to speculate that 7{beta}-hydroxycholesterol is further metabolized by sterol 27-hydroxylase to 27-hydroxy-7{beta}-hydroxycholesterol. In addition to 27-hydroxy-7KC, Brown et al. observe a large unidentified peak in macrophages incubated with 7KC (14). This peak had a retention time that was very close to a peak assigned to 7{beta}-hydroxycholesterol that was observed in sterol 27-hydroxylase-deficient mice, where as expected 27-hydroxy-7KC was absent. If the unidentified peak is indeed 7{beta}-hydroxycholesterol, one might postulate that the efficient metabolism of 7KC includes, first, the conversion to 7{beta}-hydroxycholesterol by 11{beta}-HSD1, then to 27-hydroxy-7{beta}-hydroxycholesterol by sterol 27-hydroxylase, followed by further metabolism to bile acids. In patients with cerebrotendinous xanthomatosis, lacking 27-hydroxylase, inefficient metabolism may lead to accumulation of 7{beta}-hydroxycholesterol, causing end-product inhibition of 11{beta}-HSD1, which may explain the increased 7KC concentrations in these patients.

Under normal conditions, plasma 7KC concentrations are in the nanomolar range, and metabolites of 7KC are expected to be low. However, in certain disease states or upon intake of high amounts of oxidized cholesterol-rich food, 7KC concentrations increase, and its metabolism is essential to cope with the toxic effects of 7KC. The results from our in vitro and in vivo experiments provide evidence for an important role of 11{beta}-HSD1 in the initial step of the detoxification of 7KC by the stereospecific reduction of 7KC to 7{beta}-hydroxycholesterol. Moreover, the present findings may have important implications for the appropriate application of specific 11{beta}-HSD1 inhibitors in the therapeutic treatment of obese and diabetic patients. To avoid accumulation of 7KC taken up from food, a pharmaceutical compound acting specifically on 11{beta}-HSD1 in adipose tissue may be of advantage.


   FOOTNOTES
 
* This work was supported by Swiss National Science Foundation Grants 3100AO-100060 and NRP50-4050-066575. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} A Cloëtta Research Fellow. To whom correspondence should be addressed: Div. of Nephrology and Hypertension, Dept. of Clinical Research, University of Berne, Freiburgstrasse 15, 3010 Berne, Switzerland. Tel.: 41-31-632-9438; Fax: 41-31-632-9444; E-mail: alex.odermatt{at}dkf2.unibe.ch.

1 The abbreviations used are: 7KC, 7-ketocholesterol, 3{beta}-hydroxy-5-cholesten-7-one; 11{beta}-HSD, 11{beta}-hydroxysteroid dehydrogenase; 7{beta}-hydroxycholesterol, 5-cholesten-3{beta},7{beta}-diol; 7{alpha}-hydroxycholesterol, 5-cholesten-3{beta},7{alpha}-diol; 27-hydroxycholesterol, 5-cholesten-3{beta},27{alpha}-diol; CBX, carbenoxolone; HEK-293 cells, human embryonic kidney-293 cells; GC-MS, gas chromatography-mass spectrometry. Back

2 A. Odermatt and M. Zürcher, unpublished data. Back

3 A. Odermatt, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Monika Ledermann for advice in the preparation of hepatocytes, Felipe Lopez for help with animal experimentation, and Heidi Jamin for technical assistance.



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