help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-2325
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
92/3/857    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tomlinson, J. W.
Right arrow Articles by Stewart, P. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tomlinson, J. W.
Right arrow Articles by Stewart, P. M.
Related Collections
Right arrow Adrenal and Hypertension
Right arrow Metabolism
The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 3 857-864
Copyright © 2007 by The Endocrine Society

Inhibition of 11ß-Hydroxysteroid Dehydrogenase Type 1 Activity in Vivo Limits Glucocorticoid Exposure to Human Adipose Tissue and Decreases Lipolysis

Jeremy W. Tomlinson, Mark Sherlock, Beverley Hughes, Susan V. Hughes, Fiona Kilvington, William Bartlett, Rachel Courtney, Paul Rejto, William Carley and Paul M. Stewart

Division of Medical Sciences (J.W.T., M.S., B.H., S.V.H., P.M.S.), Institute of Biomedical Research, University of Birmingham, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TT, United Kingdom; Department of Clinical Chemistry and Immunology (F.K., W.B.), Heart of England National Health Service Foundation Trust, Birmingham B9 5SS, United Kingdom; and Pfizer Global Research and Development (R.C., P.R., W.C.), La Jolla Laboratories, San Diego, California 92121

Address all correspondence and requests for reprints to: Dr. Jeremy W. Tomlinson, Ph.D., M.R.C.P., Institute of Biomedical Research, Division of Medical Sciences, University of Birmingham, Queen Elizabeth Hospital, Birmingham B15 2TT, United Kingdom. E-mail: j.w.tomlinson{at}bham.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: The pathophysiological importance of glucocorticoids (GCs) is exemplified by patients with Cushing’s syndrome who develop hypertension, obesity, and insulin resistance. At a cellular level, availability of GCs to the glucocorticoid and mineralocorticoid receptors is controlled by the isoforms of 11ß-hydroxysteroid dehydrogenase (11ß-HSD). In liver and adipose tissue, 11ß-HSD1 converts endogenous, inactive cortisone to active cortisol but also catalyzes the bioactivation of the synthetic prednisone to prednisolone.

Objective: The objective of the study was to compare markers of 11ß-HSD1 activity and demonstrate that inhibition of 11ß-HSD1 activity limits glucocorticoid availability to adipose tissue.

Design and Setting: This was a clinical study.

Patients: Seven healthy male volunteers participated in the study.

Intervention: Intervention included carbenoxolone (CBX) single dose (100 mg) and 72 hr of continuous treatment (300 mg/d).

Main Outcome Measures: Inhibition of 11ß-HSD1 was monitored using five different mechanistic biomarkers (serum cortisol and prednisolone generation, urinary corticosteroid metabolite analysis by gas chromatography/mass spectrometry, and adipose tissue microdialysis examining cortisol generation and glucocorticoid-mediated glycerol release).

Results: Each biomarker demonstrated reduced 11ß-HSD1 activity after CBX administration. After both a single dose and 72 hr of treatment with CBX, cortisol and prednisolone generation decreased as did the urinary tetrahydrocortisol+5{alpha}-tetrahydrocortisol to tetrahydrocortisone ratio. Using adipose tissue microdialysis, we observed decreased interstitial fluid cortisol availability with CBX treatment. Furthermore, a functional consequence of 11ß-HSD1 inhibition was observed, namely decreased prednisone-induced glycerol release into adipose tissue interstitial fluid indicative of inhibition of GC-mediated lipolysis.

Conclusion: CBX is able to inhibit rapidly the generation of active GC in human adipose tissue. Importantly, limiting GC availability in vivo has functional consequences including decreased glycerol release.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
THE SIMILARITIES BETWEEN patients with cortisol excess, Cushing’s syndrome, and those with obesity highlight the important role that glucocorticoids (GCs) play in the control of body composition and metabolism. However, obesity is not a state of circulating cortisol excess (1). GC availability to bind to the glucocorticoid and mineralocorticoid receptors (GRs and MRs, respectively) is controlled by the isoenzymes of 11ß-hydroxysteroid dehydrogenase (11ß-HSD). 11ß-HSD2 is located in mineralocorticoid target tissues (kidney, placenta) and inactivates cortisol to cortisone and protects the MR from occupation by cortisol for which it shares similar affinity as aldosterone. 11ß-HSD1 is located in key GC target tissues (liver, adipose, and muscle) and is a bidirectional, endolumenal enzyme that in vivo acts predominantly as an oxoreductase-generating active cortisol from inactive cortisone and thus amplifies GC action locally (2). Activity is regulated by factors including proinflammatory cytokines and growth factors (3), is crucially dependent on cofactor (nicotinamide adenine dinucleotide phosphate reduced) availability (4), and is dysregulated in obesity (5). Although levels of expression and activity of 11ß-HSD1 are still debated in the literature (6, 7, 8, 9), the most fundamental observation is that selective 11ß-HSD1 inhibition (inhibition of 11ß-HSD1 and not 11ß-HSD2) improves glucose tolerance and insulin sensitivity in rodent models (10, 11, 12). However, currently these compounds are not available for use in clinical studies. Licorice derivatives, glycyrrhizic acid, and its hydrolytic product, glycyrrhetinic acid (GE) are potent inhibitors of both 11ß-HSD1 and -2 (13, 14), causing hypertension and hypokalemia as a consequence of impaired inactivation of cortisol (inhibition of 11ß-HSD2), allowing MR activation. The derivative of GE, carbenoxolone (CBX) also inhibits 11ß-HSD1 and -2. It has been used in several clinical studies and improves whole-body insulin sensitivity and decreases glucose production rates (15, 16). However, concern has been expressed that it may not be able to access adipose tissue, and this has important implications for selective 11ß-HSD1 inhibition as a therapeutic strategy in humans (17).

GCs have potent effects on adipocytes, promoting differentiation and inhibiting omental preadipocyte proliferation (7) and induction of lipolysis (18). Whilst the mechanism of action has not been completely defined, GCs probably act to increase hormone-sensitive lipase activity, hydrolyzing triacylglycerol to diacylglycerol with the release of free fatty acids (FFAs) and glycerol (18). 11ß-HSD1 is more highly expressed in omental preadipocytes, compared with sc, and it is believed to have a fundamental role to promote adipocyte differentiation and limit preadipocyte proliferation (7, 19). Overexpression specifically within adipose tissue recapitulates many of the features of the metabolic syndrome including central obesity, hypertension, and dyslipidemia (20). Similarly, overexpression specifically within the liver causes features of the metabolic syndrome without obesity (21). Mice with targeted deletion of HSD11B1 resist diet-induced obesity and develop relative insulin sensitivity (22). More recently transgenic animals have been created that overexpress 11ß-HSD2, which is not normally expressed in adipose tissue; local inactivation of GCs by this mechanism protects against the adverse metabolic consequences of diet-induced obesity (23).

We conducted a detailed clinical study to directly compare markers of 11ß-HSD1 inhibition in vivo. In addition, using the nonselective inhibitor CBX, we investigated whether 11ß-HSD1 inhibition can limit the availability of both cortisol and prednisolone (derived from the synthetic, inactive GC, prednisone, that is dependent on 11ß-HSD1 reductase activity to generate bioactive prednisolone) in serum and adipose tissue interstitial fluid. Furthermore, we hypothesized that decreasing GC availability through 11ß-HSD1 inhibition has functional consequences to limit adipose tissue lipolysis.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
The study was approved by local research ethics committee, and all subjects gave informed consent. Seven healthy male volunteers (mean age 28 ± 3 yr, mean body mass index 24.5 ± 1.3kg/m2) were recruited and underwent two separate weeks of investigation (Fig. 1Go). Each week of investigation was separated by a washout period of at least 8 wk.


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 1. Clinical study protocol. Wk 1 and 2 protocols were performed in seven healthy adult male volunteers who were on no regular medications and had not received GC therapy within the last 12 months. Wk 1 and 2 of the protocol were separated by at least 8 wk, and in the final week of this washout period, a repeat 24-h urine collection was performed off all medication (*, administration of 10 mg prednisone, wk 1; or 25 mg cortisone acetate, wk 2).

 
Clinical protocol

Wk 1. The aims of wk 1 were to demonstrate that inhibition of 11ß-HSD1 limits prednisolone generation in serum and adipose tissue and assess the functional impact of this on adipose tissue lipolysis. Subjects were fasted and at 0830 h, an adipose tissue microdialysis catheter (CMA60; CMA microdialysis, Stockholm, Sweden) was inserted into sc adipose tissue under local anesthetic 10 cm lateral to the umbilicus. After a flush sequence (15 µl/min for 5 min), microdialysis was performed at a rate of 0.5 µl/min and continued for 30 min before sample collection (commenced at 0900 h, hourly aliquots collected for 5 h). Baseline blood samples were taken at 1000 h (t = 0 min) and 10 mg prednisone (Aventis Pharma Ltd., Paris, France) was administered orally. Blood samples were then taken at t = 20, 40, 60, 80, 100, 120, 140, 160, 180, and 240 min for biochemical analysis. On the following day, subjects were reinvestigated as described above, 2 h after a single 100-mg dose of CBX (Tokiwa Phytochemicals, Chiba, Japan) taken at 0800 h (2 hr before the cortisol generation profile) and additionally after 72 h of CBX treatment (100 mg three times per day, 0800, 1400, and 2200 h). To confirm inhibition of 11ß-HSD activity, 24-h urine collections were performed during the second day of CBX treatment (24–48 h), analyzed by gas chromatography/mass spectrometry (GC/MS) (see below) and compared with samples collected off all glucocorticoid and CBX treatment (at least 8 wk). Blood pressure was measured on each day of the protocol (average of three readings, measured supine after 10 min rest using Dynamap; Critikon, Tampa, FL).

Wk 2. To compare prednisolone generation with the more recognized marker of 11ß-HSD1 activity, cortisol generation from oral cortisone, and show that inhibition of 11ß-HSD1 activity can limit GC availability to adipose tissue, subjects were readmitted to the research facility after an 8-wk washout period. Subjects were dexamethasone suppressed [1 mg dexamethasone at 2300 h the preceding night and a further dose (0.5 mg) at 0900 h], and at 0830 h in the fasted state, a CMA60 microdialysis catheter was inserted and microdialysis performed as described above. Blood samples were again taken at 1000 h (t = 0 min) and an oral dose of 25 mg cortisone acetate (Aventis Pharma Ltd., West Malling, UK) administered. Blood samples were then taken at the following times: t = 20, 40, 60, 80, 100, 120, 140, 160, 180, 240 min. As in wk 1, investigations were repeated 2 h after a single dose and after 72 h continuous treatment with CBX. A 24-h urine collection for corticosteroid metabolite was again taken on the second day of CBX treatments and analyzed by GC/MS.

Biochemical assays

Serum. Blood counts, urea, creatinine and electrolytes, cholesterol, triglycerides, liver chemistry, and plasma glucose were measured using standard laboratory methods (Roche modular system; Roche Ltd., Lewes, UK). Serum cortisol was assayed using a chemiluminescent immunoassay (Bayer Advia Centaur; Bayer Diagnostics, Newbury, UK) with interassay coefficients of variation of 10.2% at 76 nmol/liter, 7.7% at 528 nmol/liter, and 7.4% at 882 nmol/liter. Cortisone was assayed after extraction from serum followed by RIA of the extract with 125I-cortisone and Sac-Cel (IDS Ltd., Tyne and Weir, UK) second antibody separation. The coefficient of variation for 10 consecutive assays was less than 15% for values between 50 and 100 nmol/liter and less than 10% for values over 100 nmol/liter. Prednisolone levels were analyzed by HPLC as described previously (24). Full resolution of all steroids at absorption at 254 nm was achieved. Using this assay, coefficient of variation for within-day estimates of imprecision were 4.4%. FFAs were measured in serum by the acyl-CoA synthase and acyl-CoA oxidase methods (Wako Chemicals, Neuss, Germany). Dehydroepiandrosterone was measured using a commercially available conventional two-site RIA (Diagnostic Systems Laboratories Inc., Webster, TX) and dehydroepiandrosterone sulfate measured using a coat-a-count RIA (Diagnostic Products Corp., Los Angeles, CA). Both assays were performed as per the manufacturers’ guidelines.

Stock solutions of CBX (molecular weight 446.36) and working solutions of internal standards (PF-00603176) (0.25 µg/ml) were prepared in methanol and stored at –20 C. Calibration standards (5–2500 ng/ml) and quality control (QC) samples (10, 300, 1500 ng/ml) were prepared by spiking 5 µl of the appropriate standard or QC working solutions into 50 µl of blank plasma. Aliquots of 0.15 ml acetonitrile to methanol (1:1) and 10 µl of working internal standards solution were added to all standards, QC samples, and 50 µl of unknown samples. Samples were vortexed for 2 min and then centrifuged at 1900 x g (3600 rpm) for 10 min at 5 C. Ten microliters of the supernatant from each tube were then injected into a Sciex API 4000 mass spectrometer equipped with an HPLC system (1100 binary pump; Agilent, Berks, UK) using a Synergi 4µ Hydro-RP column (30 x 2.0 mm) (Phenomenex, Torrance, CA). Due to the limitations in sample volume obtained from microdialysis, for analysis of CBX levels only, hourly samples from individuals across the GC generation profiles were pooled. Pooled samples from individuals were analyzed separately. The lower limit of detection of the assay for these samples was set at 1.0 ng/ml (2.2 nM).

Microdialysate

Microdialysate samples were collected in microvials and exchanged hourly. Cortisol was measured using a commercially available colorimetric competitive ELISA (R & D Systems, Minneapolis, MN). The minimum detectable dose range for the assay was 0.1–0.3 nmol/liter with intraassay coefficients of variation of 6–9%. Due to the semipermeable membrane within the microdialysis probe (20 kDa cutoff), microdialysate cortisol measurements reflect free, unbound cortisol in contrast to the measurements obtained in serum that are total cortisol (bound and unbound).

Samples from wk 1 were analyzed using a mobile photometric, enzyme-kinetic analyzer (CMA 600) for concentrations of glucose, pyruvate, lactate, and glycerol. CBX in microdialysate samples was measured by an HPLC method as described above.

Urinary corticosteroid metabolites

Urinary corticosteroid metabolite analysis was performed by GC/MS as described previously (25). The sum of total cortisol metabolites [THF (tetrahydrocortisol), THE (tetrahydrocortisone), 5{alpha}THF, {alpha}-cortolone, cortisone, cortisol, ß-cortolone, ß-cortol, {alpha}-cortol] provides a reflection of cortisol secretion rate. More specifically, the ratio of tetrahydrometabolites of cortisol (THF + 5{alpha}THF) to those of cortisone (THE) provides a reflection of 11ß-HSD1 activity when considered with the ratio of urinary free cortisol (UFF) to cortisone (UFE), which more accurately reflects renal 11ß-HSD2 activity. The activities of 5{alpha}- and 5ß-reductases can be inferred from measuring the ratio of THF to 5{alpha}THF.

Statistical analysis

Power and sample size calculations were performed and based on predicted changes in urinary steroid metabolite ratios. Data are presented as mean ± SE unless otherwise stated. Area under the curve (AUC) analysis was performed using the trapezoidal method. For comparison of single variables between wk 1 and 2 of the clinical protocol, paired t tests have been used. Where repeated samples have been taken, repeated-measures ANOVA has been used. All analysis was performed using the SigmaStat 3.1 software package (Systat Software, Inc., Point Richmond, CA).


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Clinical parameters before and during carbenoxolone treatment are presented in Table 1Go and urinary corticosteroid metabolites in Table 2Go.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Clinical, anthropometric, and biochemical characterization of seven healthy male subjects before, 2 h after a single dose of CBX (100 mg), and after 72 h of CBX treatment (300 mg/d)

 

View this table:
[in this window]
[in a new window]

  		TABLE 2. Urinary corticosteroid metabolite analysis performed by GC/MS on 24-h urine samples from seven healthy male volunteers before and after 48 h continuous CBX treatment (300 mg/d)

 
Urine collections on CBX were performed 24–48 h after the administration of glucocorticoid and therefore are unlikely to have been influenced by cortisone, dexamethasone, or prednisone administration. Total cortisol metabolite production decreased after CBX administration. Consistent with inhibition of 11ß-HSD2, UFF to UFE ratio increased significantly. Despite this, the THF+5{alpha}THF to THE ratio decreased after CBX treatment as did the ratio of cortols to cortolones consistent with inhibition of 11ß-HSD1 activity. There was no alteration in the activities of 5{alpha}- or 5ß-reductase. Results of the urinary GC/MS analysis after CBX treatment were similar in wk 1 and 2.

Prednisolone and cortisol generation

After oral administration of prednisone (10 mg) or cortisone acetate (25 mg), all subjects generated prednisolone and cortisol, respectively (Fig. 2Go). A single dose of CBX decreased prednisolone generation [AUC: 1671 ± 64 (baseline) vs. 1491 ± 61 nmol/liter·h (CBX), P < 0.05]; however, cortisol generation was not different [AUC: 1617 ± 125 (baseline) vs. 1499 ± 103 nmol/liter·h (CBX), P = not significant (ns)]. After 72 h CBX treatment, inhibition of prednisolone generation was maintained (AUC: 1443 ± 80 nmol/liter·h, P < 0.05 vs. baseline), but cortisol generation decreased significantly (AUC: 1203 ± 76 nmol/liter·h, P < 0.05 vs. baseline) (Fig. 2Go). Peak prednisolone concentrations did not decrease after a single dose of CBX (574 ± 26 vs. 554 ± 42 nmol/liter, P = ns) but fell after 72 h of CBX administration (475 ± 24 nmol/liter, P < 0.05). Similarly, a single dose of CBX did not affect peak cortisol concentrations, but these fell after 72 h of CBX treatment [peak cortisol: 579 ± 25 (baseline) vs. 546 ± 33 (single dose CBX), P = ns, 465 ± 21 nmol/liter (72 h CBX), P < 0.05].


Figure 2
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 2. Cortisol generation after an oral dose of cortisone acetate (25 mg) does not change after a single dose of CBX, 100 mg (gray triangles, hashed bars). However, after 72 h treatment, 300 mg/d (filled squares and bars) cortisol generation significantly decreased [generation profile (A) and AUC analysis (B)]. Prednisolone generation form oral prednisone (10 mg) decreases after both a single dose (100 mg) and 72 h (300 mg/d) treatment with CBX [generation profile (C) and AUC analysis (D)] (baseline samples, white circles and bars).

 
Cortisone concentrations after dexamethasone suppression were undetectable and increased after oral cortisone acetate (36 ± 13, t = 20 min; 74 ± 10, t = 40 min; 72 ± 10, t = 60 min; 78 ± 7, t = 120 min; 57 ± 4 nmol/liter, t = 240 min). Cortisone appearance in the serum was similar before and after single dose of CBX [AUC cortisone: 241 ± 27 vs. 223 ± 27 nmol/liter·h (single dose CBX), P = ns] but decreased after 72 h of treatment [167 ± 19 nmol/liter·h, P < 0.005 vs. baseline and P < 0.01 vs. single dose], consistent with inhibition of renal 11ß-HSD2 limiting cortisone regeneration from cortisol.

Adipose tissue interstitial fluid cortisol concentrations were low but detectable within the limits of the assay after dexamethasone suppression (0.5 ± 0.2 nmol/liter). After oral cortisone acetate, cortisol concentrations in adipose tissue interstitial fluid increased (Fig. 3AGo). Both a single dose and 72 h of treatment with CBX decreased cortisol concentrations after oral cortisone within adipose tissue interstitial fluid [peak cortisol: 8.9 ± 2.1 (baseline), 5.6 ± 1.1 (single dose CBX), 5.3 ± 0.6 nmol/liter (72 h CBX, P = 0.07]. In addition, AUC for cortisol generation fell significantly, indicative of inhibition of 11ß-HSD1 activity (Fig. 3Go, A and B).


Figure 3
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 3. A single dose of CBX, 100 mg (gray triangles, hashed bars), and 72 h treatment, 300 mg/d (filled squares and bars) limit cortisol availability within adipose tissue interstitial fluid after oral cortisone acetate, 25 mg as measured by microdialysis [generation profile (A) and AUC analysis (B)]. After oral prednisone, 10 mg, glycerol release into adipose tissue interstitial fluid increases (C). CBX treatment (single dose, gray triangles, hashed bars; and 72 h, filled squares and bars) decreases prednisone-induced glycerol release [glycerol release (C), AUC analysis (D)] (baseline samples, white circles and bars).

 
CBX concentration in serum and interstitial fluid

Serum CBX concentrations as measured by HPLC were detectable 2 h after a single 100-mg dose (19.6 ± 2.5 µmol/liter) but fell within 4 h (10.3 ± 1.6 µmol/liter, P < 0.005). After 72 h of CBX treatment, CBX levels increased (96.2 ± 9.4 µmol/liter, P < 0.001 vs. single dose) and showed no decrease within 4 h of a morning dose (103.6 ± 10.7 µmol/liter, P = ns). Although concentrations were greater than 5000-fold less than those observed in serum (Table 1Go), we were able to measure CBX within adipose tissue interstitial fluid. After a single dose of CBX, the concentration within adipose interstitial fluid was at the lower limit of detection of the CBX assay (2.0 ± 1.0 nmol/liter). However, after 72 h of CBX treatment, levels increased to 19.7 ± 15.0 nmol/liter. Before CBX administration, CBX levels were undetectable.

Glucocorticoid-induced lipolysis

Prednisone administration (wk 1) caused a significant rise in serum FFA levels in all subjects: (73 ± 18, t = 0 min vs. 175 ± 35 µmol/liter, t = 240 min, P < 0.05). Whilst we were unable to detect a difference in serum FFA concentrations after CBX administration (either single dose or 72 h treatment), peak prednisolone concentration (both on and off CBX) correlated positively with FFA generation (R = 0.49, P < 0.05).

Subcutaneous adipose tissue interstitial fluid glycerol concentrations increased after oral prednisone (63.0 ± 23.5, t = 0 min vs. 223.9 ± 24.0 µmol/liter, t = 240 min, P < 0.01). Both a single dose and 72 h treatment with CBX decreased glycerol concentrations after oral prednisone [peak glycerol: 240.9 ± 23.1 (baseline), 193.3 ± 17.3 (single dose CBX), P < 0.05; 174.4 ± 29.1 µmol/liter (72 h CBX), P < 0.05]. In addition, AUC for glycerol release decreased (Fig. 3Go, C and D). There were no significant changes in adipose tissue interstitial fluid concentrations of glucose, lactate, or pyruvate (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
In this paper we have shown that CBX inhibits the generation of bioactive cortisol and prednisolone through inhibition of 11ß-HSD1. Inhibition can be measured in urine (a measure of global 11ß-HSD activity) and serum (largely reflecting hepatic activity) as well as adipose tissue interstitial fluid. Finally and most importantly, decreasing GC availability is functionally important in human sc adipose tissue, limiting GC-induced lipolysis.

The changes in serum biochemistry during this study were small. However, the decrease in urea and potassium during wk 1 (and not wk 2) may reflect the additional mineralocorticoid action of prednisolone with consequent fluid retention. Additionally, in wk 1 (in contrast to wk 2), endogenous GC production was not suppressed, and therefore, CBX inhibition of renal 11ß-HSD2 would enhance mineralocorticoid action of endogenous cortisol (through inhibition of inactivation to cortisone). Although bioequivalent doses of cortisol and prednisolone have similar potencies for MR activation, the 10-mg dose of prednisone and 25 mg of cortisone acetate are not bioequivalent; indeed the former represents an approximate doubling of mineralocorticoid action. This mechanism may also contribute to the observed decrease in hemoglobin concentration (hemodilution), but the contribution of repeated blood sampling over a relatively short period of time maybe a factor.

In previous studies, CBX increased the UFF to UFE ratio by 53–102%, reflecting inhibition of renal 11ß-HSD2 (26, 27), and our data (45–62%) are consistent with this; the decrease in the THF+5{alpha}THF to THE ratio is less marked (28–34%), but again our values are comparable (32–33%). Inhibition of 11ß-HSD2 by CBX will inhibit the conversion of cortisol to cortisone, and therefore, selective 11ß-HSD1 inhibitors are likely to cause a more significant decrease in the THF+5{alpha}THF to THE ratio. Overall, the data presented validate the use of urinary GC/MS to assess 11ß-HSD1 inhibition.

11ß-HSD1 and -2 are able to interconvert cortisol and cortisone as well as prednisone and prednisolone, and activity of both enzymes is inhibited by GE (28, 29, 30). Using in vitro systems, enzyme kinetics are similar for both substrates (28). During the cortisol generation profile (wk 2), subjects have endogenous cortisol production suppressed using dexamethasone. GCs are potent up-regulators of 11ß-HSD1 activity and expression, and as a result baseline measurements of activity may be artificially high. In addition, dexamethasone has been shown to be a substrate for 11ß-HSD1 and could therefore compete with cortisone (31). CBX not only acts as a competitive inhibitor of 11ß-HSD1 but also decreases mRNA expression (32), and this may counteract the GC induction on subsequent days. Inhibition of 11ß-HSD2 prolongs cortisol and prednisolone half-life (33) and therefore would increase total cortisol and prednisolone generation. In agreement with this, we observed decreased total cortisol metabolite production after CBX treatment, suggestive of a predominant action of CBX on 11ß-HSD2; however, this could also reflect a degree of hypothalamus-pituitary-adrenal axis suppression after the administration of exogenous GC (prednisone or dexamethasone). Selective 11ß-HSD1 inhibitors would be predicted to have a more dramatic impact on these profiles in comparison with CBX, and as such the generation of both prednisolone and cortisol may be useful biomarkers.

In healthy adults, CBX improves whole-body insulin sensitivity (15), and in patients with type 2 diabetes, glucose production rates are decreased through a reduction in glycogenolysis (16). In healthy volunteers, CBX decreases intraocular pressure (26), and more recent studies have demonstrated improvements in cognitive function in elderly men and those with type 2 diabetes (34). As an agent that causes acquired apparent mineralocorticoid excess through its inhibition of 11ß-HSD2, CBX is not viable as a long-term therapeutic strategy, and much attention has now been focused on the development of selective 11ß-HSD1 inhibitors (35). Such compounds administered to rodents improve insulin sensitivity, glucose tolerance, lipid profiles, and decreased atherogenesis (10, 11, 12); however, to date there are no published data on clinical studies in humans.

Within sc adipose tissue interstitial fluid, a single dose of CBX is as effective as 72 h continuous treatment at limiting cortisol availability. Compared with the parallel serum measurements, which are believed to most accurately reflect hepatic activity (5, 36), inhibition of activity was more marked in the microdialysate samples (45 vs. 25%). Cortisol production in the microdialysate was inhibited after a single dose of CBX when serum cortisol was unaffected until 72 h of treatment. This disconnect in the inhibition measured in microdialysate and serum is consistent with local inhibition in adipose. This hypothesis is further strengthened by the observation that we were able to measure CBX within adipose tissue interstitial fluid. Although CBX concentrations in the serum were 5000-fold greater than those in adipose tissue interstitial fluid, these concentrations are still in excess of interstitial fluid GC concentrations, and therefore, functionally important competitive inhibition seems plausible. However, the current study does not allow us to categorically determine whether the changes observed in adipose tissue represent local inhibition or are a reflection of changes in circulatory GC levels. Interestingly, because adipose tissue does not express 11ß-HSD2 (37), it may also provide the cleanest system for looking at 11ß-HSD1 inhibition by CBX. A further consideration is that tissue-specific regulation of 11ß-HSD1 activity and expression is well described, and this could explain differences between some of the biomarkers measured (3).

A single previous study has used adipose microdialysis to examine cortisol metabolism in adipose tissue (17). The methodology used was different, and tritiated substrate was introduced via the microdialysis catheter. In our study, microdialysis was simply used to sample adipose tissue interstitial fluid to determine the expose of adipocytes to extracellular GCs (using this method we cannot comment on intracellular GC availability) before and after CBX treatment. Although differences were observed between lean and obese individuals in the previous study, inhibition with CBX could not be demonstrated (17). Substrate delivery, metabolism, and product recovery by this methodology is extremely complex and subject to a large number of important variables including steroid distribution within lipid and tissue blood flow. The authors’ conclusion was that CBX could not access adipose tissue interstitial fluid; however, our data suggest that this now seems unlikely.

GCs promote lipolysis (18, 38), and we have been able to show for the first time that activity of 11ß-HSD1 is a critical regulator of this process in vivo. Whereas peak prednisolone after oral prednisone correlated significantly with FFA release in to the serum, we were unable to show differences in FFA generation before or after CBX. This may well reflect rapid reesterification of released FFAs but may also reflect the fact that changes in serum prednisolone (before and after CBX) were small (albeit significant), perhaps reflecting concomitant inhibition of renal 11ß-HSD2. The adipose tissue microdialysis data, using prednisone as an 11ß-HSD1 substrate, reveal that CBX decreased peak glycerol release indicative of inhibition of GC-mediated lipolysis, although the AUC analysis achieved only a borderline significant result (P = 0.06). Unfortunately, due to the small volumes of microdialysate samples and lack of a sensitive and specific assay method, direct measurement of interstitial fluid prednisolone was not possible. The role of FFAs in the control of insulin sensitivity is well described (39), and it is possible that the improvement in insulin sensitivity in rodents treated with selective 11ß-HSD1 inhibitors may arise due to decreased adipose tissue-derived FFAs. In addition, FFAs have been reported to regulate 11ß-HSD1 in one study (40) although not in another (41).

To conclude, urinary GC/MS, serum prednisolone, and serum and interstitial adipose cortisol generation are useful biomarkers of 11ß-HSD1 inhibition. Limitation of GC availability to human adipose tissue through inhibition of 11ß-HSD1 has functional consequences including decreased lipolysis.


   Acknowledgments
 
We thank all the nursing staff (in particular Jo Finney) on the Wellcome Trust Clinical Research facility (Queen Elizabeth Hospital, Birmingham, UK) where this study took place; Penny Clarke (Regional Endocrine Laboratory, University Hospitals Birmingham National Health Service Trust, Birmingham, UK); and Sue Zhou (Pfizer Inc.) for their assistance with the serum biochemical analysis. We also thank Mike Jirousek and Boaz Hirshberg (Pfizer) for their helpful comments in the analysis of the data.


   Footnotes
 
The study was funded by the Wellcome Trust [program grant reference 066357/Z/01/Z (to P.M.S.) and clinician scientist fellowship reference 075322/Z/04/Z (to J.W.T.)], the Medical Research Council (experimental medicine initiative reference G0502165), and an investigator-initiated research grant from Pfizer.

Disclosure Statement: J.W.T., M.S., B.H., S.V.H., and F.K. have nothing to declare. W.B. is on the advisory board for Abbot Diagnostics, United Kingdom. R.C., P.R., and W.C. are employees of Pfizer Global Research and Development, and P.R. has equity interests in Pfizer. P.M.S. has a consultancy with Pfizer Global Research and Development.

First Published Online January 2, 2007

Abbreviations: AUC, Area under the curve; CBX, carbenoxolone; FFA, free fatty acid; GC, glucocorticoid; GC/MS, gas chromatography/mass spectrometry; GE, glycyrrhetinic acid; 11ß-HSD, 11ß-hydroxysteroid dehydrogenase; MR, mineralocorticoid receptor; ns, not significant; QC, quality control; THE, tetrahydrocortisone; THF, tetrahydrocortisol; UFE, urinary free cortisone; UFF, urinary free cortisol.

Received October 24, 2006.

Accepted December 22, 2006.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Fraser R, Ingram MC, Anderson NH, Morrison C, Davies E, Connell JM 1999 Cortisol effects on body mass, blood pressure, and cholesterol in the general population. Hypertension 33:1364–1368[Abstract/Free Full Text]
  2. Tomlinson JW, Walker EA, Bujalska IJ, Draper N, Lavery GG, Cooper MS, Hewison M, Stewart PM 2004 11ß-hydroxysteroid dehydrogenase type 1: a tissue-specific regulator of glucocorticoid response. Endocr Rev 25:831–866[Abstract/Free Full Text]
  3. Tomlinson JW, Moore J, Cooper MS, Bujalska I, Shahmanesh M, Burt C, Strain A, Hewison M, Stewart PM 2001 Regulation of expression of 11ß-hydroxysteroid dehydrogenase type 1 in adipose tissue: tissue-specific induction by cytokines. Endocrinology 142:1982–1989[Abstract/Free Full Text]
  4. Lavery GG, Walker EA, Draper N, Jeyasuria P, Marcos J, Shackleton CH, Parker KL, White PC, Stewart PM 2005 Hexose-6-phosphate dehydrogenase knockout mice lack 11ß-hydroxysteroid dehydrogenase type 1-mediated glucocorticoid generation. J Biol Chem 281:6546–6551
  5. Stewart PM, Boulton A, Kumar S, Clark PM, Shackleton CH 1999 Cortisol metabolism in human obesity: impaired cortisone–>cortisol conversion in subjects with central adiposity. J Clin Endocrinol Metab 84:1022–1027[Abstract/Free Full Text]
  6. Rask E, Walker BR, Soderberg S, Livingstone DE, Eliasson M, Johnson O, Andrew R, Olsson T 2002 Tissue-specific changes in peripheral cortisol metabolism in obese women: increased adipose 11ß-hydroxysteroid dehydrogenase type 1 activity. J Clin Endocrinol Metab 87:3330–3336[Abstract/Free Full Text]
  7. Tomlinson JW, Sinha B, Bujalska I, Hewison M, Stewart PM 2002 Expression of 11ß-hydroxysteroid dehydrogenase type 1 in adipose tissue is not increased in human obesity. J Clin Endocrinol Metab 87:5630–5635[Abstract/Free Full Text]
  8. Goedecke JH, Wake DJ, Levitt NS, Lambert EV, Collins MR, Morton NM, Andrew R, Seckl JR, Walker BR 2006 Glucocorticoid metabolism within superficial subcutaneous rather than visceral adipose tissue is associated with features of the metabolic syndrome in South African women. Clin Endocrinol (Oxf) 65:81–87[CrossRef][Medline]
  9. Desbriere R, Vuaroqueaux V, Achard V, Boullu-Ciocca S, Labuhn M, Dutour A, Grino M 2006 11ß-hydroxysteroid dehydrogenase type 1 mRNA is increased in both visceral and subcutaneous adipose tissue of obese patients. Obesity (Silver Spring) 14:794–798
  10. Hermanowski-Vosatka A, Balkovec JM, Cheng K, Chen HY, Hernandez M, Koo GC, Le Grand CB, Li Z, Metzger JM, Mundt SS, Noonan H, Nunes CN, Olson SH, Pikounis B, Ren N, Robertson N, Schaeffer JM, Shah K, Springer MS, Strack AM, Strowski M, Wu K, Wu T, Xiao J, Zhang BB, Wright SD, Thieringer R 2005 11ß-HSD1 inhibition ameliorates metabolic syndrome and prevents progression of atherosclerosis in mice. J Exp Med 202:517–527[Abstract/Free Full Text]
  11. Alberts P, Engblom L, Edling N, Forsgren M, Klingstrom G, Larsson C, Ronquist-Nii Y, Ohman B, Abrahmsen L 2002 Selective inhibition of 11ß-hydroxysteroid dehydrogenase type 1 decreases blood glucose concentrations in hyperglycaemic mice. Diabetologia 45:1528–1532[CrossRef][Medline]
  12. Alberts P, Nilsson C, Selen G, Engblom LO, Edling NH, Norling S, Klingstrom G, Larsson C, Forsgren M, Ashkzari M, Nilsson CE, Fiedler M, Bergqvist E, Ohman B, Bjorkstrand B, Abrahmsen LB 2003 Selective inhibition of 11ß-hydroxysteroid dehydrogenase type 1 improves hepatic insulin sensitivity in hyperglycemic mice strains. Endocrinology 144:4755–4762[Abstract/Free Full Text]
  13. Monder C, Stewart PM, Lakshmi V, Valentino R, Burt D, Edwards CRW 1989 Licorice inhibits corticosteroid 11ß-dehydrogenase of rat liver and kidney: in vivo and in vitro studies. Endocrinology 124:1046–1053
  14. Stewart PM, Murry BA, Mason JI 1994 Human kidney 11ß-hydroxysteroid dehydrogenase is a high affinity nicotinamide adenine dinucleotide-dependent enzyme and differs from the cloned type I isoform. J Clin Endocrinol Metab 79:480–484[Abstract]
  15. Walker BR, Connacher AA, Lindsay RM, Webb DJ, Edwards CR 1995 Carbenoxolone increases hepatic insulin sensitivity in man: a novel role for 11-oxosteroid reductase in enhancing glucocorticoid receptor activation. J Clin Endocrinol Metab 80:3155–3159[Abstract]
  16. Andrews RC, Rooyackers O, Walker BR 2003 Effects of the 11ß-hydroxysteroid dehydrogenase inhibitor carbenoxolone on insulin sensitivity in men with type 2 diabetes. J Clin Endocrinol Metab 88:285–291[Abstract/Free Full Text]
  17. Sandeep TC, Andrew R, Homer NZ, Andrews RC, Smith K, Walker BR 2005 Increased in vivo regeneration of cortisol in adipose tissue in human obesity and effects of the 11ß-hydroxysteroid dehydrogenase type 1 inhibitor carbenoxolone. Diabetes 54:872–879[Abstract/Free Full Text]
  18. Slavin BG, Ong JM, Kern PA 1994 Hormonal regulation of hormone-sensitive lipase activity and mRNA levels in isolated rat adipocytes. J Lipid Res 35:1535–1541[Abstract]
  19. Bujalska IJ, Kumar S, Hewison M, Stewart PM 1999 Differentiation of adipose stromal cells: the roles of glucocorticoids and 11ß-hydroxysteroid dehydrogenase. Endocrinology 140:3188–3196[Abstract/Free Full Text]
  20. Masuzaki H, Yamamoto H, Kenyon CJ, Elmquist JK, Morton NM, Paterson JM, Shinyama H, Sharp MG, Fleming S, Mullins JJ, Seckl JR, Flier JS 2003 Transgenic amplification of glucocorticoid action in adipose tissue causes high blood pressure in mice. J Clin Invest 112:83–90[CrossRef][Medline]
  21. Paterson JM, Morton NM, Fievet C, Kenyon CJ, Holmes MC, Staels B, Seckl JR, Mullins JJ 2004 Metabolic syndrome without obesity: hepatic overexpression of 11ß-hydroxysteroid dehydrogenase type 1 in transgenic mice. Proc Natl Acad Sci USA 101:7088–7093[Abstract/Free Full Text]
  22. Kotelevtsev Y, Holmes MC, Burchell A, Houston PM, Schmoll D, Jamieson P, Best R, Brown R, Edwards CR, Seckl JR, Mullins JJ 1997 11ß-Hydroxysteroid dehydrogenase type 1 knockout mice show attenuated glucocorticoid-inducible responses and resist hyperglycemia on obesity or stress. Proc Natl Acad Sci USA 94:14924–14929[Abstract/Free Full Text]
  23. Kershaw EE, Morton NM, Dhillon H, Ramage L, Seckl JR, Flier JS 2005 Adipocyte-specific glucocorticoid inactivation protects against diet-induced obesity. Diabetes 54:1023–1031[Abstract/Free Full Text]
  24. Cooper MS, Blumsohn A, Goddard PE, Bartlett WA, Shackleton CH, Eastell R, Hewison M, Stewart PM 2003 11ß-Hydroxysteroid dehydrogenase type 1 activity predicts the effects of glucocorticoids on bone. J Clin Endocrinol Metab 88:3874–3877[Abstract/Free Full Text]
  25. Palermo M, Shackleton CH, Mantero F, Stewart PM 1996 Urinary free cortisone and the assessment of 11ß-hydroxysteroid dehydrogenase activity in man. Clin Endocrinol (Oxf) 45:605–611[CrossRef][Medline]
  26. Rauz S, Cheung CM, Wood PJ, Coca-Prados M, Walker EA, Murray PI, Stewart PM 2003 Inhibition of 11ß-hydroxysteroid dehydrogenase type 1 lowers intraocular pressure in patients with ocular hypertension. QJM 96:481–490[Abstract/Free Full Text]
  27. Andrew R, Smith K, Jones GC, Walker BR 2002 Distinguishing the activities of 11ß-hydroxysteroid dehydrogenases in vivo using isotopically labeled cortisol. J Clin Endocrinol Metab 87:277–285[Abstract/Free Full Text]
  28. Cooper MS, Rabbitt EH, Goddard PE, Bartlett WA, Hewison M, Stewart PM 2002 Osteoblastic 11ß-hydroxysteroid dehydrogenase type 1 activity increases with age and glucocorticoid exposure. J Bone Miner Res 17:979–986[CrossRef][Medline]
  29. Hundertmark S, Buhler H, Rudolf M, Weitzel HK, Ragosch V 1997 Inhibition of 11ß-hydroxysteroid dehydrogenase activity enhances the antiproliferative effect of glucocorticosteroids on MCF-7 and ZR-75–1 breast cancer cells. J Endocrinol 155:171–180[Abstract]
  30. Chen MF, Shimada F, Kato H, Yano S, Kanaoka M 1990 Effect of glycyrrhizin on the pharmacokinetics of prednisolone following low dosage of prednisolone hemisuccinate. Endocrinol Jpn 37:331–341[Medline]
  31. Diederich S, Eigendorff E, Burkhardt P, Quinkler M, Bumke-Vogt C, Rochel M, Seidelmann D, Esperling P, Oelkers W, Bahr V 2002 11ß-Hydroxysteroid dehydrogenase types 1 and 2: an important pharmacokinetic determinant for the activity of synthetic mineralo- and glucocorticoids. J Clin Endocrinol Metab 87:5695–5701[Abstract/Free Full Text]
  32. Whorwood CB, Sheppard MC, Stewart PM 1993 Licorice inhibits 11ß-hydroxysteroid dehydrogenase messenger ribonucleic acid levels and potentiates glucocorticoid hormone action. Endocrinology 132:2287–2292[Abstract]
  33. Stewart PM, Wallace AM, Atherden SM, Shearing C, Edwards CRW 1990 Mineralocorticoid activity of carbenoxolone: contrasting effects of carbenoxolone and liquorice on 11ß-hydroxysteroid dehydrogenase activity in man. Clin Sci 78:49–54
  34. Sandeep TC, Yau JL, MacLullich AM, Noble J, Deary IJ, Walker BR, Seckl JR 2004 11ß-Hydroxysteroid dehydrogenase inhibition improves cognitive function in healthy elderly men and type 2 diabetics. Proc Natl Acad Sci USA 101:6734–6739[Abstract/Free Full Text]
  35. Tomlinson JW, Stewart PM 2005 Mechanisms of disease: selective 11-HSD1 inhibition as a novel treatment for the metabolic syndrome. Nat Clin Pract Endocrinol Metab 1:92–99[CrossRef][Medline]
  36. Rask E, Olsson T, Soderberg S, Andrew R, Livingstone DE, Johnson O, Walker BR 2001 Tissue-specific dysregulation of cortisol metabolism in human obesity. J Clin Endocrinol Metab 86:1418–1421[Abstract/Free Full Text]
  37. Bujalska IJ, Kumar S, Stewart PM 1997 Does central obesity reflect "Cushing’s disease of the omentum"? Lancet 349:1210–1213[CrossRef][Medline]
  38. Djurhuus CB, Gravholt CH, Nielsen S, Mengel A, Christiansen JS, Schmitz OE, Moller N 2002 Effects of cortisol on lipolysis and regional interstitial glycerol levels in humans. Am J Physiol Endocrinol Metab 283:E172–E177
  39. Kovacs P, Stumvoll M 2005 Fatty acids and insulin resistance in muscle and liver. Best Pract Res Clin Endocrinol Metab 19:625–635[CrossRef][Medline]
  40. Wake DJ, Homer NZ, Andrew R, Walker BR 2006 Acute in vivo regulation of 11ß-hydroxysteroid dehydrogenase type 1 activity by insulin and Intralipid infusions in humans. J Clin Endocrinol Metab 91:4682–4688[Abstract/Free Full Text]
  41. Mai K, Bobbert T, Kullmann V, Andres J, Bahr V, Maser-Gluth C, Rochlitz H, Spranger J, Diederich S, Pfeiffer AF 2006 No effect of free fatty acids on adrenocorticotropin and cortisol secretion in healthy young men. Metabolism 55:1022–1028[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J Mol EndocrinolHome page
Y. Liu, Y. Nakagawa, Y. Wang, L. Liu, H. Du, W. Wang, X. Ren, K. Lutfy, and T. C Friedman
Reduction of hepatic glucocorticoid receptor and hexose-6-phosphate dehydrogenase expression ameliorates diet-induced obesity and insulin resistance in mice
J. Mol. Endocrinol., August 1, 2008; 41(2): 53 - 64.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
J. W. Tomlinson, J. Finney, B. A. Hughes, S. V. Hughes, and P. M. Stewart
Reduced Glucocorticoid Production Rate, Decreased 5{alpha}-Reductase Activity, and Adipose Tissue Insulin Sensitization After Weight Loss
Diabetes, June 1, 2008; 57(6): 1536 - 1543.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
D. P Macfarlane, S. Forbes, and B. R Walker
Glucocorticoids and fatty acid metabolism in humans: fuelling fat redistribution in the metabolic syndrome
J. Endocrinol., May 1, 2008; 197(2): 189 - 204.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
I. J. Bujalska, K. N. Hewitt, D. Hauton, G. G. Lavery, J. W. Tomlinson, E. A. Walker, and P. M. Stewart
Lack of Hexose-6-Phosphate Dehydrogenase Impairs Lipid Mobilization from Mouse Adipose Tissue
Endocrinology, May 1, 2008; 149(5): 2584 - 2591.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
R. Courtney, P. M. Stewart, M. Toh, M.-N. Ndongo, R. A. Calle, and B. Hirshberg
Modulation of 11{beta}-Hydroxysteroid Dehydrogenase (11{beta}HSD) Activity Biomarkers and Pharmacokinetics of PF-00915275, a Selective 11{beta}HSD1 Inhibitor
J. Clin. Endocrinol. Metab., February 1, 2008; 93(2): 550 - 556.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
B. G. Bhat, N. Hosea, A. Fanjul, J. Herrera, J. Chapman, F. Thalacker, P. M. Stewart, and P. A. Rejto
Demonstration of Proof of Mechanism and Pharmacokinetics and Pharmacodynamic Relationship with 4'-Cyano-biphenyl-4-sulfonic Acid (6-Amino-pyridin-2-yl)-amide (PF-915275), an Inhibitor of 11 -Hydroxysteroid Dehydrogenase Type 1, in Cynomolgus Monkeys
J. Pharmacol. Exp. Ther., January 1, 2008; 324(1): 299 - 305.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
A. M. Nuotio-Antar, D. L. Hachey, and A. H. Hasty
Carbenoxolone treatment attenuates symptoms of metabolic syndrome and atherogenesis in obese, hyperlipidemic mice
Am J Physiol Endocrinol Metab, December 1, 2007; 293(6): E1517 - E1528.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
S. A. Wudy, M. F. Hartmann, and T. Remer
Sexual dimorphism in cortisol secretion starts after age 10 in healthy children: urinary cortisol metabolite excretion rates during growth
Am J Physiol Endocrinol Metab, October 1, 2007; 293(4): E970 - E976.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
92/3/857    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tomlinson, J. W.
Right arrow Articles by Stewart, P. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tomlinson, J. W.
Right arrow Articles by Stewart, P. M.
Related Collections
Right arrow Adrenal and Hypertension
Right arrow Metabolism

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals