A combination of polymorphisms in HSD11B1 associates with in vivo 11β-HSD1 activity and metabolic syndrome in women with and without polycystic ovary syndrome

in European Journal of Endocrinology
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  • 1 Division of Endocrinology, Endocrinology Unit, Division of Internal Medicine, Department of Clinical Medicine, Centre for Applied Biomedical Research (CRBA), S. Orsola‐Malpighi Hospital, University of Bologna – Alma Mater Studiorum, Via Massarenti 9, 40138 Bologna, Italy

Objective

Regeneration of cortisol by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) within liver and adipose tissue may be of pathophysiological importance in obesity and the metabolic syndrome. single nucleotide polymorphisms (SNPs) in HSD11B1, the gene encoding 11β-HSD1, have been associated with type 2 diabetes and hypertension in population-based cohort studies, and with hyperandrogenism in patients with the polycystic ovary syndrome (PCOS). However, the functional consequences of these SNPs for in vivo 11β-HSD1 expression and activity are unknown.

Methods

We explored associations of well-characterised hormonal and metabolic phenotypes with two common SNPs (rs846910 and rs12086634) in HSD11B1 in 600 women (300 with PCOS) and investigated 11β-HSD1 expression and activity in a nested study of 40 women from this cohort.

Results

HSD11B1 genotypes (as single SNPs and as the combination of the two minor allele SNPs) were not associated with PCOS. Women who were heterozygous for rs846910 A and homozygous for rs12086634 T (GA, TT genotype) had a higher risk of metabolic syndrome, regardless of the diagnosis of PCOS (odds ratio in the whole cohort=2.77 (95% confidence interval (CI) 1.16–6.67), P=0.023). In the nested cohort, women with the GA, TT genotype had higher HSD11B1 mRNA levels in adipose tissue, and higher rates of appearance of cortisol and d3-cortisol (16.1±0.7 nmol/min versus 12.1±1.1, P=0.044) during 9,11,12,12-2H4-cortisol (d4-cortisol) steady-state infusion.

Conclusions

We conclude that, in a population of Southern European Caucasian women with and without PCOS, alleles of HSD11B1 containing the two SNPs rs846910 A and rs12086634 T confer increased 11β-HSD1 expression and activity, which associates with the metabolic syndrome.

Abstract

Objective

Regeneration of cortisol by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) within liver and adipose tissue may be of pathophysiological importance in obesity and the metabolic syndrome. single nucleotide polymorphisms (SNPs) in HSD11B1, the gene encoding 11β-HSD1, have been associated with type 2 diabetes and hypertension in population-based cohort studies, and with hyperandrogenism in patients with the polycystic ovary syndrome (PCOS). However, the functional consequences of these SNPs for in vivo 11β-HSD1 expression and activity are unknown.

Methods

We explored associations of well-characterised hormonal and metabolic phenotypes with two common SNPs (rs846910 and rs12086634) in HSD11B1 in 600 women (300 with PCOS) and investigated 11β-HSD1 expression and activity in a nested study of 40 women from this cohort.

Results

HSD11B1 genotypes (as single SNPs and as the combination of the two minor allele SNPs) were not associated with PCOS. Women who were heterozygous for rs846910 A and homozygous for rs12086634 T (GA, TT genotype) had a higher risk of metabolic syndrome, regardless of the diagnosis of PCOS (odds ratio in the whole cohort=2.77 (95% confidence interval (CI) 1.16–6.67), P=0.023). In the nested cohort, women with the GA, TT genotype had higher HSD11B1 mRNA levels in adipose tissue, and higher rates of appearance of cortisol and d3-cortisol (16.1±0.7 nmol/min versus 12.1±1.1, P=0.044) during 9,11,12,12-2H4-cortisol (d4-cortisol) steady-state infusion.

Conclusions

We conclude that, in a population of Southern European Caucasian women with and without PCOS, alleles of HSD11B1 containing the two SNPs rs846910 A and rs12086634 T confer increased 11β-HSD1 expression and activity, which associates with the metabolic syndrome.

Introduction

HSD11B1(*600713) encodes the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which catalyses regeneration of cortisol from its inactive metabolite cortisone, thereby amplifying glucocorticoid receptor activation, e.g. in liver and adipose tissue (1).

Both increased and decreased 11β-HSD1 activity have been implicated in the pathophysiology of common diseases. Glucocorticoid excess, e.g. in Cushing's syndrome, can cause features of the metabolic syndrome, including central obesity, hypertension and glucose intolerance. Transgenic overexpression of 11β-HSD1 in liver (2) or adipose tissue (3) in mice increases local glucocorticoid concentrations and recapitulates these features of metabolic syndrome. Conversely, inhibition (4, 5, 6) or disruption (7, 8, 9) of 11β-HSD1 ameliorates features of the metabolic syndrome. In human obesity, 11β-HSD1 expression is increased in adipose tissue (5, 10) but simultaneously decreased in liver (10, 11). Decreased 11β-HSD1 expression results in impaired regeneration of cortisol and hence increased metabolic clearance rate for cortisol; the resulting compensatory activation of the hypothalamic–pituitary–adrenal axis may be responsible for adrenal androgen excess in some patients with polycystic ovary syndrome (PCOS) (12, 13, 14).

Although a variety of hormonal and nutritional factors regulate 11β-HSD1 expression (1, 5), there is circumstantial evidence that genetic factors contribute to inter-individual variation in cortisol regeneration. Common non-coding single nucleotide polymorphisms (SNPs) in the 5′-flanking region of HSD11B1 (rs846910, G to A) and in an enhancer region in intron 3 (rs12086634, T to G) have been associated independently with insulin resistance (15), type 2 diabetes (15) and/or hypertension (16, 17) in several studies, although these observations have not been reproduced in all populations (18) and these variants are not associated with obesity. Conversely, the G allele of rs12086634, which causes lower 11β-HSD1 transcriptional activity in vitro (14), is associated with hyperandrogenism among lean women with PCOS (13), although it is not more common among PCOS cases as a whole (19). However, the consequences of these non-coding polymorphisms for 11β-HSD1 expression and function in vivo have not been determined.

In subcutaneous adipose tissue biopsies, neither rs846910 nor rs12086634 genotype predicted HSD11B1 mRNA in adipocytes in 61 subjects from a native N American cohort (15). Urinary ratios of cortisol:cortisone metabolites, an indicator of activity of steroid metabolising enzymes including 11β-HSD1, are not associated with rs846910 genotype (20, 21).

We performed this study to address the following research questions: i) does the distribution of rs846910 and rs12086634 gene variants differ between PCOS and controls? ii) are rs846910 A and rs12086634 T genotypes associated with the metabolic syndrome? iii) is this eventual association a characteristic of PCOS or is it present in the whole cohort? iv) do rs846910 and rs12086634 gene variants influence in vivo 11β-HSD1 expression and activity?

To answer these questions, we genotyped a cohort of 600 women in whom metabolic and endocrine variables were carefully assessed and half of whom had PCOS, and selected 40 participants with contrasting genotypes for a nested study in which we performed more detailed measurements of 11β-HSD1 activity, including stable isotope tracer measurements (22).

Methods

Participants and protocols for the main cohort

We investigated 300 unmedicated Caucasian women with PCOS, aged 18–45 years, and 300 Caucasian controls recruited from the general population in Northern Italy, and compared for age and body weight. PCOS women had at least two of the following characteristics: i) chronic oligoanovulation; ii) hirsutism (Ferriman–Gallwey score ≥8 or elevated serum total testosterone levels (23)); iii) polycystic ovarian morphology at ultrasound, according to the Rotterdam consensus conference criteria (24).

Hyperprolactinaemia, Cushing's syndrome, congenital adrenal hyperplasia and androgen-secreting tumors were excluded by laboratory analysis (23). Inclusion criteria for controls were the absence of signs of hyperandrogenism (hirsutism, acne or alopecia) and the presence of regular ovulatory menstrual cycles (progesterone levels ≥8 ng/ml during the luteal phase (23)). Exclusion criteria for both PCOS and controls were the presence of thyroid dysfunction, cardiovascular, renal or liver diseases screened by clinical examination and routine laboratory findings. Anthropometric data (height, weight, waist and hip circumferences), and systolic and diastolic blood pressure were measured, as described previously (23). All subjects completed a 24 h urine collection and attended our centre at 0800–0830 h after overnight fast. Basal blood samples for metabolic measurements (glucose, insulin, total cholesterol, HDL-cholesterol and triglycerides) were collected. Samples were immediately chilled on ice and centrifuged, and serum was stored at −20 °C. Blood samples for DNA extraction were collected in disodium-EDTA and stored at 4 °C. Studies were performed between day 5 and 10 of the menstrual cycle, or during amenorrhoea, after excluding pregnancy by appropriate testing.

The metabolic syndrome was assessed by the National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (NCEP/ATPIII) criteria (25), as we have recently demonstrated the high concordance between the three classifications (NCEP/ATPIII, International Diabetes Federation (IDF) and American Heart Association/National Heart Lung and Blood Institute (AHA/NHLBI)) in diagnosing the metabolic syndrome in PCOS women as well as in controls (26).

NCEP-ATPIII criteria required that an individual meet at least three of the following parameters: i) waist circumference ≥88 cm; ii) fasting glucose ≥110 mg/dl (6.105 mmol/l); iii) triglycerides ≥150mg/dl (1.695 mmol/l); iv) HDL-cholesterol <50 mg/dl (1.295 mmol/l) and v) blood pressure ≥130/85 mmHg.

Participants and protocols for the nested cohort

From the main cohort, 40 women were selected to provide approximately balanced groups of subjects with contrasting HSD11B1 genotypes and balanced numbers with or without PCOS within each genotype group. We first randomly selected PCOS subjects from each genotype group and then chose controls matched to each PCOS subject for genotype, age, body weight and waist circumference. These women attended our centre on two further study days. On the first day, they completed a 24 h urine collection for the measurement of cortisol and its metabolites, and attended after overnight fast. To evaluate adipose 11β-HSD1 expression and activity, ∼300 mg subcutaneous needle aspiration biopsy of subcutaneous adipose tissue from the anterior abdominal wall were obtained and stored at −80 °C as described previously (27). After the biopsy procedure, 9,11,12,12-(2H)4-cortisol (d4-cortisol, Cambridge Isotopes, Andover, MA, USA) was infused at 20 molar per cent excess in cortisol (Flebocortid Richer, Sanofi-Aventis S.p.A., Milano, Italy) at 1.74 mg/h after a priming bolus dose of 3.5 mg, and blood sampled at intervals for 4 h (22). d4-Cortisol was administered for estimating whole-body regeneration of cortisol by 11β-HSD1. The evening prior to the second assessment day, subjects took an oral dose of 1 mg dexamethasone (Decadron, Visufarma S.p.A., Roma, Italy) at 2300 h, for suppressing the endogenous cortisol production, and fasted overnight. At 0800 h, an oral dose of 25 mg cortisone acetate (Cortone Acetato, Giuseppe Rende S.r.L., Roma, Italy) was administered, and blood samples were obtained at intervals for 4 h to measure plasma cortisol. The conversion of oral cortisone into plasma cortisol allowed us to estimate the activity of 11β-HSD1 in the liver.

Approval from the ethics committee and informed consent was obtained for all studies, according to the guidelines established by the Institutional Review Board at S. Orsola-Malpighi Hospital and in accordance with the Declaration of Helsinki.

Genotyping

DNA was extracted using QIAamp DNA Blood Kit (Qiagen, Inc.). Genotyping of rs12086634 and rs846910 was undertaken by single nucleotide primer extension (SNuPE) and denaturing high-performance liquid chromatography (DHPLC), adapted from Hoogendoorn et al. (28). Both HSD11B1 gene fragments were amplified together by PCR for 35 cycles, each consisting of 30 s at 95 °C, 30 s at 65 °C and 20 s at 72 °C. Primers for rs12086634 were 5′-CTGAGGTTTGCCCAACAAGATTTC-3′ (forward) and 5′-CACTGCTGGAGGTGAGTATTAGAG-3′ (reverse), and for rs846910, 5′-GCAGCCTCAGCACACTACATTG-3′ (forward) and 5′-GTCCCACTTACCAGCCAGAGAG-3′ (reverse). Reactions were performed in 10 μl final volume using 25 pmol of each primer, 40 ng of genomic DNA, 200 μM dNTPs, 2 mM MgCl2 and 0.08 units of AmpliTaq Gold (Applied Biosystems) in the buffer provided by the manufacturer. PCR products were treated with 2 μl Exo-SAP-IT (Amersham Biosciences) to hydrolyse unincorporated nucleotides and degrade primers in excess. Primer extension reactions were carried out in one reaction in 25 μl containing the purified PCR products, 50 μM of the appropriate ddNTPs (ddATP, ddGTP and ddCTP), 200 pmol primer and 1.25 units of Thermo Sequenase (Amersham Biosciences) in the buffer provided by the manufacturer. SNuPE reactions were performed in a thermal cycler with 35 cycles each consisting of 30 s at 94 °C, 15 s at 55 °C, 60 s at 60 °C, followed by 2 s at 15 °C. Primers were forward 5′-GTTGCTTGTGCTTGATTCCATTTATTCTGGTG-3′ for rs12086634 and reverse 5′-GCAAGAGATGGCTATATTAAGAAATC-3′ for rs846910. Solution (10 μl) containing extended primers was loaded on a SaraSep DNASep column (Transgenomic, San José, CA, USA) at 80 °C and separated by DHPLC (Wave, Transgenomic) using a linear acetonitrile gradient (over 7 min from 20 to 37% acetonitrile in 0.1 M triethylamine acetate buffer, pH 7) at a constant flow rate of 0.9 ml/min. Data were acquired using a u.v. detector at 260 nm.

To confirm the effectiveness of the method, 100 samples were previously screened by allelic discrimination TaqMan Assay by design (Applied Biosystems) for rs12086634 and by direct sequencing using Big Dye Terminator for rs846910, as described by Nair et al. (15).

Hormone and biochemical assays

The assays for hormonal and biochemical measurements have been reported elsewhere (23, 29, 30).

Analyses of d4-cortisol and its metabolites

Plasma 9,11,12,12-(2H)4-cortisol (d4-cortisol), 9,12,12-(2H)3-cortisone (d3-cortisone) and 9,12,12-(2H)3-cortisol (d3-cortisol) were measured by liquid chromatography/tandem mass spectrometry as described previously (31), and tracer kinetics was calculated using the mean of five measurements in steady state between 180 and 240 min of d4-cortisol infusion. Rate of appearance of d3-cortisol was calculated as (d4-cortisol infusion rate)/(d4-cortisol:d3-cortisol ratio). Rate of appearance of cortisol was calculated as (d4-cortisol infusion rate)/(d4-cortisol:cortisol ratio)−(cortisol infusion rate).

Analysis of urinary steroids

Cortisol and its metabolites (5β-tetrahydrocortisol (5β-THF), 5α-THF, 5β-tetrahydrocortisone (5β-THE), cortols, cortolones and cortisone) were measured in 24 h urine by electron impact gas chromatography–mass spectrometry (GC–MS) with minor modifications from a previously described method (32). Briefly, an urine aliquot was equilibrated with internal standards (11α-epi-THF and 11α-hydrocortisone). Steroids were purified by Sep-Pak C18 extraction, and conjugates were hydrolysed with β-glucuronidase, re-extracted and converted to methoxyamine–trimethylsilylimidazole derivatives, before injection into a GC–MS (Agilent, Santa Clara, CA, USA: GC 6890–MS 5973) in selected ion mode. Cortisol and its metabolites were quantified by the ratio of metabolite:internal standard area against standard curves for each steroid, included in every assay batch. Total cortisol excretion was calculated from the sum of 5β-THF, 5α-THF, 5β-THE, cortols and cortolones. The balance of 11β-HSD1 and 11β-HSD2 activities in all tissues was assessed as the ratio (5α-THF+5β-THF)/5β-THE, and renal 11β-HSD2 activity was assessed as urinary cortisol/cortisone ratio.

Analysis of adipose tissue 11β-HSD1 mRNA and protein levels

Approximately, 500 mg of fat were homogenised in 1 ml TRIzol (Invitrogen), RNA quantified by spectrophotometry, and RNA integrity checked by agarose gel electrophoresis. RNA (1 μg) was reverse transcribed using the Invitrogen Reverse Transcription System, and transcript levels for 11β-HSD1 were quantified by real-time PCR with primer–probe sets from PE Applied Biosystems: 5′-GGAATATTCAGTGTCCAGGGTCAA-3′ (forward); 5′-TGATCTCCAGGGCACATTCCT-3′ (reverse), and 5′-6-FAM-CTTGGCCTCATAGACACAGAAACAGCCA-TAMRA-3′ (probe). Amplification and detection were performed with the iCycler iQ Real-Time PCR detection system (Bio-Rad Laboratories) with the following profile: 50 cycles consisting of 30 s at 95 °C and 60 s at 60 °C. The real-time PCR amplifications were performed in 25 μl reaction volume with 300 mM primers, 300 mM TaqMan probe and 2×IQ Supermix (Bio-Rad Laboratories). Human cyclophilin A (PE Applied Biosystems) was used to normalise the 11β-HSD1 transcript levels. A standard curve was generated in triplicate by serial dilution of cDNA pooled from several subjects. Each sample was run in duplicate, and the mean of the duplicates was expressed as a fold difference in RNA level versus an internal control sample. RT negative controls and intron spanning primers were used to control for genomic DNA contamination and prevent its amplification respectively.

To estimate total 11β-HSD1 protein, activity was measured in the dehydrogenase direction, which is the most stable in vitro, as described previously (10). Briefly, 250 mg tissue were homogenised in KREBS buffer. Total protein (400 μg/ml) was incubated with 2 mM NADP, 0.2% glucose and 100 nM cortisol (of which 10 nM 1,2,6,7-(3H)4-cortisol) and incubated at 37 °C for 30 h. Aliquots were withdrawn at intervals (6, 24 and 30 h), and conversion to 1,2,6,7-(3H)4-cortisone was measured by HPLC with online scintillation detection.

Results are expressed as picomole of product generated per microgram of homogenised tissue.

Statistical analysis

The number of cases included in the main cohort was calculated taking into account the previously reported minor allele frequency (MAF; A allele) for SNP rs846910 of 13% in PCOS and of 6% in controls (13); a case–control study of 600 subjects has a power of 80% to replicate this difference in prevalence at a significance level of P<0.05. The number of cases included in the nested cohort was calculated anticipating a difference between genotype groups of at least 20% in d3-cortisol production and 5% in 11β-HSD1 mRNA level; a case–control study comparing groups of ten subjects has a power of 80% to detect these differences at a significance level of P<0.05.

The distribution of HSD11B1 genotypes (rs846910 and rs12086634) as single SNPs and as the combination of the two SNPs in PCOS cases versus controls was analysed by the χ2 test. It was not possible to classify haplotypes for rs846910 and rs12086634 accurately because of the ambiguity estimated for the haplotypes GT/AG or GG/AT in either PCOS or controls by Arlequin analysis. Whether the distribution of genotypes was consistent with the Hardy–Weinberg equilibrium was examined using the χ2 test, and linkage disequilibrium was measured by Haploview 4.1 Software (http://www.broad.mit.edu/mpg/haploview).

Continuous data were evaluated by one-way or two-way ANOVA, or two-way repeated-measures ANOVA, as appropriate. Bivariate logistic regression was used to estimate the association of the metabolic syndrome or of the individual components of the metabolic syndrome with HSD11B1 genotypes (as a combination of rs846910 and rs12086634) and with PCOS status.

Data are shown as mean±s.e.m. and frequencies. Statistical analyses were performed by the SPSS/PC+ version 8 Software package (Chicago, IL, USA). Two-tailed P values <0.05 were considered statistically significant.

Results

Analyses in 600 women with and without PCOS

The distributions of genotypes for single SNPs and for the combination of the two SNPs in the whole cohort are shown in Table 1. MAF were 0.038 for rs846910 A allele and 0.151 for rs12086634 G allele. No participants were homozygous for rs846910 A allele. The two SNPs did not deviate from the Hardy–Weinberg equilibrium in either group (P=0.51 and 0.47) and did not show linkage disequilibrium (D′=0.21 and r2=0.01). There were no differences in the prevalence of either SNP or combination of the two SNPs between PCOS cases and controls. Very few participants had genotypes GA, GG (n=1) or GG, GG (n=11) so they were included in the GA, TG and GG, TG groups, respectively, in further analyses.

Table 1

Genotype distribution of HSD11B1 single nucleotide polymorphisms (SNPs; rs846910 and rs12086634) shown as single SNP and as the combination of the two SNPs in 300 polycystic ovary syndrome (PCOS) and 300 controls.

GenotypePCOSn (%)Controlsn (%)P*
rs8469100.760
 GG276 (92)278 (93)
 GA24 (8)22 (7)
 AA0 (0)0 (0)
rs120866340.920
 TT216 (72)215 (72)
 TG78 (26)79 (26)
 GG6 (2)6 (2)
rs846910, rs12086634a
 GG, TT199 (66.3)206 (68.7)0.410
 GG, TG72 (24)66 (22)
 GG, GG5 (1.7)6 (2)
 GA, TT17 (5.7)9 (3)
 GA, TG6 (2)13 (4.3)
 GA, GG1 (0.3)0 (0)

*P values were calculated by Pearson's χ2.

Genotype is shown as alleles for rs846910 followed by alleles for rs12086634.

There was a significant association between GA, TT genotype and risk of the metabolic syndrome (odds ratio (OR) 2.77, P=0.023) in the whole cohort, regardless of the diagnosis of PCOS (Table 2). In contrast, the genotype GG, TG or GG, GG was protective against the metabolic syndrome in the whole cohort (OR=0.43, P=0.011). There were no interactions between genotype and PCOS status in predicting the metabolic syndrome (Table 2).

Table 2

Association between HSD11B1 genotype (as combination of SNPs rs846910 and rs12086634) and the metabolic syndrome by National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults criteria in PCOS cases and controls, and in the whole cohort.

PCOSControlsWhole cohort
Genotypean (%)OR (95% CI)P*n (%)OR (95% CI)P*n (%)OR (95% CI)P*
GG, TT45/199 (22.6)1.0 (ref)34/206 (16.5)1.0 (ref)79/405 (19.5)1.0 (ref)
GG, TG  or GG12/77 (15.6)0.63 (0.32–1.27)0.1994/72 (5.6)0.30 (0.10–0.87)0.02716/149 (10.7)0.43 (0.23–0.82)0.011
GA, TT8/17 (47.1)3.04 (1.11–8.34)0.0313/9 (33.3)2.53 (0.60–10.61)0.20511/26 (42.3)2.77 (1.16–6.67)0.023
GA, TG  or GG2/7 (28.6)1.37 (0.26–7.30)0.7130/13 (0)0.001 (0.000–0.026)0.6952/20 (10)0.07 (0.00–15.82)0.606

*P values were calculated by logistic regression; the interaction between PCOS status and genotype did not enter into the analysis. Metabolic syndrome was associated with PCOS status, regardless of genotype (PCOS=67/300, 22.3% versus controls=41/300, 13.7%; OR (95% CI)=1.82 (1.19–2.78); P value by logistic regression adjusted for genotype=0.006.

Genotype is shown as alleles for rs846910 followed by alleles for rs12086634.

Regarding individual components of the metabolic syndrome, we found that GA, TT genotype was significantly associated with low HDL-cholesterol (OR=3.20, P=0.011) and with hypertension (OR=2.60, P=0.031), whereas GG, TG or GG, GG genotype was protective against high waist circumference (OR=0.55, P=0.005) in the whole cohort, regardless of the diagnosis of PCOS (Table 3). There was no association detected between HSD11B1 genotypes and either high glucose or high triglycerides (Table 3).

Table 3

Association between HSD11B1 genotype (as combination of SNPs rs846910 and rs12086634) and individual components of the metabolic syndrome in PCOS cases and controls, and in the whole cohort.

PCOSControlsWhole cohort
Association of genotypean (%)OR (95% CI)P*n (%)OR (95% CI)P*n (%)OR (95% CI)P*
With high waist
 GG, TT97/199 (48.7)1.0 (ref)77/206 (37.4)1.0 (ref)174/405 (43.0)1.0 (ref)
 GG, TG or GG32/77 (41.6)0.75 (0.44–1.27)0.28414/72 (19.4)0.40 (0.21–0.77)0.00646/149 (30.9)0.55 (0.37–0.84)0.005
 GA, TT10/17 (58.8)1.50 (0.55–4.10)0.4285/9 (55.5)2.09 (0.55–8.04)0.28115/26 (57.7)1.77 (0.77–4.11)0.181
 GA, TG or GG4/7 (57.1)1.40 (0.31–6.43)0.6644/13 (30.8)0.74 (0.22–2.50)0.6338/20 (40)1.02 (0.39–2.70)0.965
With high glucose
 GG, TT9/199 (4.5)1.0 (ref)8/206 (3.9)1.0 (ref)17/405 (4.2)1.0 (ref)
 GG, TG or GG3/77 (3.9)0.86 (0.23–3.25)0.8193/72 (4.2)1.08 (0.28–4.17)0.9166/149 (4.0)0.96 (0.37–2.48)0.932
 GA, TT1/17 (5.9)1.32 (0.16–11.08)0.7990/9 (0.0)0.00 (0.00–0.05)0.8571/26 (3.8)0.06 (0.00–0.08)0.863
 GA, TG or GG0/7 (0.0)0.01 (0.00–0.02)0.8220/13 (0.0)0.00 (0.00–0.08)0.8280/20 (0.0)0.00 (0.00–0.02)0.795
With high triglycerides
 GG, TT13/199 (6.5)1.0 (ref)27/206 (13.1)1.0 (ref)40/405 (9.9)1.0 (ref)
 GG, TG or GG7/77 (9.1)1.43 (0.55–3.73)0.4655/72 (6.9)0.50 (0.18–1.34)0.16612/149 (8.1)0.84 (0.42–1.68)0.624
 GA, TT2/17 (11.8)1.91 (0.39–9.25)0.4230/9 (0.0)0.00 (0.00–0.01)0.8262/26 (7.7)0.06 (0.00–22.40)0.779
 GA, TG or GG1/7 (14.3)2.38 (0.27–21.31)0.4370/13 (0.0)0.00 (0.00–0.02)0.7911/20 (5.0)0.07 (0.00–92.90)0.746
With low HDL-C
 GG, TT74/199 (37.2)1.0 (ref)34/206 (16.5)1.0 (ref)108/405 (26.7)1.0 (ref)
 GG, TG or GG27/77 (35.1)0.91 (0.53–1.58)0.74310/72 (13.9)0.82 (0.38–1.75)0.60137/149 (24.8)0.86 (0.54–1.38)0.538
 GA, TT12/17 (70.6)4.05 (1.37–11.96)0.0113/9 (33.3)2.53 (0.60–10.61)0.20515/26 (57.7)3.20 (1.30–7.86)0.011
 GA, TG or GG3/7 (42.8)1.27 (0.28–5.82)0.7611/13 (7.7)0.42 (0.05–3.35)0.4144/20 (20.0)0.73 (0.20–2.65)0.633
With hypertension
 GG, TT88/199 (44.2)1.0 (ref)61/206 (29.6)1.0 (ref)149/405 (36.8)1.0 (ref)
 GG, TG or GG30/77 (39.0)0.81 (0.47–1.38)0.42920/72 (27.8)0.91 (0.50–1.66)0.76950/149 (33.6)0.86 (0.57–1.28)0.454
 GA, TT9/17 (52.9)1.42 (0.53–3.83)0.4906/9 (66.7)4.75 (1.15–19.63)0.03115/26 (57.7)2.60 (1.09–6.17)0.031
 GA, TG or GG3/7 (42.9)0.95 (0.21–4.34)0.9432/13 (15.4)0.43 (0.09–2.01)0.2855/20 (25)0.64 (0.22–1.89)0.418

*P values were calculated by logistic regression; the interaction between PCOS status and genotype did not enter into the analysis. High waist, low HDL-cholesterol and hypertension were associated with PCOS status, regardless of genotype (high waist, PCOS=143/300, 47.7% versus controls=100/300, 33.3%; OR (95% CI)=1.82 (1.31–2.53); P value by logistic regression adjusted for genotype <0.001; low HDL-cholesterol, PCOS=116/300, 38.7% versus controls=48/300, 16.0%; OR (95% CI)=3.30 (2.25–4.87); P value by logistic regression adjusted for genotype <0.001; hypertension, PCOS=130/300, 43.3% versus controls=89/300, 29.7%; OR (95% CI)=1.81 (1.29–2.54); P value by logistic regression adjusted for genotype=0.005). No significant associations were found between high glucose or high triglycerides and PCOS status.

Genotype is shown as alleles for rs846910 followed by alleles for rs12086634.

Cortisol metabolites were measured in urine to assess relationships between HSD11B1 genotypes and in vivo 11β-HSD1 activity (Table 4). In accordance with previous reports for individual SNPs (22, 23), the combination of rs846910 and rs12086634 SNPs did not predict urinary (5α-THF+5β-THF)/5β-THE ratio.

Table 4

24 h urinary cortisol metabolites in PCOS cases and controls according to HSD11B1 genotypes (as combination of SNPs rs846910 and rs12086634). Data are mean±s.e.m. Genotypes are shown as alleles for rs846910 followed by alleles for rs12086634.

VariablesGG, TT (n=405)GG, TG or GG (n=149)GA, TT (n=26)GA, TG or GG (n=20)P for genotypes*P for diagnosis*P for interaction*
Cortisol (μg/days)
 PCOS101±498±8137±21140±230.1190.2020.556
 Controls140±4129±590±4215±25
Cortisone (μg/days)
 PCOS171±6170±10172±30237±600.2160.3440.843
 Controls 224±8192±10206±21308±68
5β-Tetrahydrocortisol (5β-THF, μg/day)
 PCOS1681±651711±1233266±6202042±4490.1450.8500.424
 Controls2173±611491±871823±3402207±20
5α-THF (μg/days)
 PCOS2404±1172110±2383900±7403539±9840.9360.3390.345
 Controls2579±1222526±2371613±1652238±106
5β-Tetrahydrocortisone (5β-THE, μg/day)
 PCOS3098±1183492±2023268±7262974±5510.9860.7850.605
 Controls3543±122931±1233543±583534±183
Total (μg/days)
 PCOS11191±37311458±71014706±196613722±28740.5750.9410.709
 Controls12979±39510211±50812619±84712854±496
Cortisol/cortisone
 PCOS0.639±0.0230.593±0.0270.843±0.0610.671±0.1180.8650.6600.734
 Controls0.716±0.0200.732±0.0270.471±0.0700.874±0.113
(5α-THF+5β-THF)/5β-THE
 PCOS1.63±0.111.08±0.053.60±1.152.56±1.170.5230.1950.510
 Controls1.65±0.141.34±0.060.98±0.161.27±0.04

*P values were calculated by two-way ANOVA; P for genotypes, comparison among the four genotypes; P for diagnosis, comparison between PCOS and controls; P for interaction, evaluation of the interaction between genotypes and diagnosis in the entire population.

Analyses in 40 women with contrasting HSD11B1 genotypes

To characterise the effect of HSD11B1 genotypes on in vivo 11β-HSD1 expression and activity in greater detail, a nested study was conducted in 20 PCOS and 20 control women representing the four most common genotypes (GA, TT, n=4 PCOS and n=4 controls; GG, TT, n=6 PCOS and n=6 controls; GA, TG, n=4 PCOS and n=4 controls; GG, TG, n=6 PCOS and n=6 controls). The group with the GA, TT genotype had higher whole body rates of appearance of both cortisol (reflecting the combination of adrenal cortisol secretion and net regeneration of cortisol by 11β-HSD1) and d3-cortisol (reflecting exclusively the contribution of 11β-HSD1 (22)) (Fig. 1A and B). In addition, women with the GA, TT genotype had higher adipose 11β-HSD1 mRNA levels (Fig. 1C). The activity of 11β-HSD1 in the liver (Fig. 2A), measured as appearance of cortisol on first pass conversion after an oral dose of cortisone, and in subcutaneous adipose tissue (Fig. 2B) was not significantly different in women with different genotypes.

Figure 1
Figure 1

Whole-body 11β-HSD1 activity, measured by d4-cortisol infusion test, and adipose 11β-HSD1 expression according to HSD11B1 genotype (as combination of SNPs rs846910 and rs12086634). Data are mean±s.e.m. for women with genotypes GA, TT (n=4 PCOS, n=4 controls); GG, TT (n=6 PCOS, n=6 controls); GA, TG (n=4 PCOS, n=4 controls); and GG, TG (n=6 PCOS, n=6 controls). (A) Rate of appearance of cortisol calculated from dilution of d4-cortisol by unlabelled cortisol during steady-state d4-cortisol infusion. By one-way ANOVA, genotype had an effect (P=0.040). (B) Rate of appearance of d3-cortisol calculated from dilution of d4-cortisol by d3-cortisol during steady-state d4-cortisol infusion. By one-way ANOVA, genotype had an effect (P=0.044). (C) 11β-HSD1 mRNA levels in subcutaneous adipose tissue, expressed as a ratio to cyclophilin internal control. By one-way ANOVA, genotype had an effect (P=0.041). *P<0.05, **P<0.01 versus GA, TT.

Citation: European Journal of Endocrinology 165, 2; 10.1530/EJE-11-0091

Figure 2
Figure 2

Subcutaneous adipose tissue and liver 11β-HSD1 activity according to HSD11B1 genotype (as combination of SNPs rs846910 and rs12086634). Data are mean±s.e.m. for women with genotypes GA, TT (n=4 PCOS, n=4 controls); GG, TT (n=6 PCOS, n=6 controls); GA, TG (n=4 PCOS, n=4 controls); and GG, TG (n=6 PCOS, n=6 controls). By two-way repeated-measures ANOVA, genotype did not have an effect on activity time course in subcutaneous adipose tissue (P=0.553) or liver (P=0.998).

Citation: European Journal of Endocrinology 165, 2; 10.1530/EJE-11-0091

Discussion

In this study, we found that: i) the distribution of rs846910 and rs12086634 gene variants does not differ between PCOS and controls; ii) rs846910 A and rs12086634 T genotype is associated with the metabolic syndrome; iii) this association was evident in the whole cohort; and iv) rs846910 A and rs12086634 T genotype is characterised by increased 11β-HSD1 transcript levels in adipose tissue and increased whole-body regeneration of cortisol from cortisone.

Therefore, these results show that Southern European Caucasian women with HSD11B1 alleles containing the two SNPs rs846910 A and rs12086634 T have increased adipose 11β-HSD1 expression and increased whole-body 11β-HSD1 activity, associated with increased prevalence of the metabolic syndrome. These findings strengthen the evidence that variations in 11β-HSD1 activity influence the metabolic profile and provide a crucial missing link by demonstrating that HSD11B1 genotypes influence enzyme activity in vivo.

Previous studies of variation in the HSD11B1 gene in native N American Pima Indians showed that rs846910 and rs12086634 are in complete linkage disequilibrium and independently associate with type 2 diabetes and hypertension (15, 16). The discrepancy with our results, where we found that the two SNPs do not show linkage disequilibrium, probably depends on the type of population studied. In particular, Pima Indians are genetically homogenous in contrast to Caucasian women, studied by us, who have a great genetic admixture. The allele frequencies in Pima Indians were higher (rs846910 A 13%; rs12086634 T 46%), and there was no interaction in that population to suggest that combination of genotypes is important. However, studies in European populations have focused on variants in intron 3 closely linked to rs12086634 (14) and have not found associations with features of metabolic syndrome (18). Our data suggest that, at least in Europeans, alterations associated with the intron 3 enhancer region (14) are insufficient to measurably affect phenotype.

Moreover, a variation in the 5′-flanking region associated with rs846910 is insufficient to alter enzyme activity, consistent with our recent findings that the in vitro HSD11B1 promoter activity is the same for both alleles of rs846910, irrespective of the addition of the transcriptional activator, C/EBPα (33). However, in this population, the combination of variation in intron 3 and in the 5′-flanking region is sufficient to increase 11β-HSD1 expression and activity.

This is the first study to undertake comprehensive functional studies of 11β-HSD1 activity, both whole body and tissue specific, in subjects with contrasting HSD11B1 genotypes. Ratios of cortisol/cortisone metabolites in urine provide an accessible, but imprecise, index of whole-body 11β-HSD1 activity, because these ratios are influenced by the activity of other enzymes, such as 11β-HSD2 and reductases. Similar to other reports (20, 21), we did not find any relationship between HSD11B1 genotypes and urinary cortisol/cortisone metabolite ratios (Table 4). Whole-body regeneration of cortisol by 11β-HSD1 can be measured specifically using the d4-cortisol stable isotope tracer (22, 34, 35). The rate of appearance of d3-cortisol was strikingly higher in women with the GA, TT HSD11B1 genotype. The major source of extra-adrenal cortisol regeneration is the liver (34, 35), but recent data confirm that there is a contribution from adipose tissue (31).

Liver 11β-HSD1 can be measured without hepatic vein cannulation by administering oral cortisone and measuring the initial rate of appearance of cortisol in peripheral plasma after first pass hepatic metabolism (35). Using this tool, we did not find an association between HSD11B1 genotype and liver 11β-HSD1 activity. However, in subcutaneous adipose tissue, women with the GA, TT genotype had higher 11β-HSD1 transcript levels. Tissue-specific dysregulation of 11β-HSD1 is well established in obesity in rodents and humans (1, 10), and may relate in part to tissue-specific use of promoters (36) and/or to differential effects of C/EBP transcription factors in liver and adipose (37); the promoter activity may be influenced by genetic variation.

There is good evidence that genetic variation in the gene encoding hexose-6-phosphate dehydrogenase (H6PDH) influences 11β-HSD1 activity by controlling availability of cofactor NADPH within the endoplasmic reticulum (14, 38, 39). Profound deficiency in 11β-HSD1 activity appears to result from mutations in H6PDH rather than HSD11B1 (14). However, we did not investigate the role of H6PDH in this study, because mutations in H6PDH are too rare to obtain adequate statistical power in this cohort.

Genetic association studies must be interpreted cautiously in view of the risk of false positives. However, several observations suggest that the associations of HSD11B1 genotype with metabolic syndrome observed in this study are not spurious. Variants in HSD11B1 have been associated with the metabolic syndrome in other cohorts (15, 16, 17) but are not included in the chips that have been used commonly in genome-wide association studies to date. We have shown that HSD11B1 genotype predicts variation in enzyme function, and we know from numerous studies in animal models and in humans that variations in 11β-HSD1 activity influence metabolic syndrome variables (1). The association of HSD11B1 genotypes with the metabolic syndrome was driven primarily by links with low HDL-cholesterol and high blood pressure; HSD11B1 genotype was not closely linked with glucose homeostasis or hypertriglyceridaemia. In mice, increased 11β-HSD1 activity selectively in liver (2) or adipose tissue (3) is sufficient to induce hypertension and hyperinsulinaemia, while cross-sectional studies in humans describe associations of adipose 11β-HSD1 expression with insulin sensitivity and blood pressure (40). Although the relative youth of this cohort of women may mitigate against detecting effects on glucose, this observation may be important in highlighting different consequences of variations in 11β-HSD1 in mice and humans.

Our results confirm previous reports that variants in HSD11B1 are not associated with the prevalence of PCOS (19, 20) and extend these findings to include rs846910 and its combination with rs12086634.

In conclusion, complex variations in the HSD11B1 gene have functional consequences for enzyme activity in vivo, particularly in adipose tissue, which influence fat distribution and its metabolic consequences. These findings reinforce the importance of 11β-HSD1 as a pathophysiological mediator and therapeutic target in the metabolic syndrome.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Acknowledgements

We thank Vincenzo Cerreta, Jill Harrison and Alison Rutter for their assistance and technical advice, and the staff of the Wellcome Trust Clinical Research Facility Mass Spectrometry Core Laboratory for their technical support. We also thank Fondazione Cassa di Risparmio in Bologna for supporting the Centre for Applied Biomedical Research.

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    Whole-body 11β-HSD1 activity, measured by d4-cortisol infusion test, and adipose 11β-HSD1 expression according to HSD11B1 genotype (as combination of SNPs rs846910 and rs12086634). Data are mean±s.e.m. for women with genotypes GA, TT (n=4 PCOS, n=4 controls); GG, TT (n=6 PCOS, n=6 controls); GA, TG (n=4 PCOS, n=4 controls); and GG, TG (n=6 PCOS, n=6 controls). (A) Rate of appearance of cortisol calculated from dilution of d4-cortisol by unlabelled cortisol during steady-state d4-cortisol infusion. By one-way ANOVA, genotype had an effect (P=0.040). (B) Rate of appearance of d3-cortisol calculated from dilution of d4-cortisol by d3-cortisol during steady-state d4-cortisol infusion. By one-way ANOVA, genotype had an effect (P=0.044). (C) 11β-HSD1 mRNA levels in subcutaneous adipose tissue, expressed as a ratio to cyclophilin internal control. By one-way ANOVA, genotype had an effect (P=0.041). *P<0.05, **P<0.01 versus GA, TT.

  • View in gallery

    Subcutaneous adipose tissue and liver 11β-HSD1 activity according to HSD11B1 genotype (as combination of SNPs rs846910 and rs12086634). Data are mean±s.e.m. for women with genotypes GA, TT (n=4 PCOS, n=4 controls); GG, TT (n=6 PCOS, n=6 controls); GA, TG (n=4 PCOS, n=4 controls); and GG, TG (n=6 PCOS, n=6 controls). By two-way repeated-measures ANOVA, genotype did not have an effect on activity time course in subcutaneous adipose tissue (P=0.553) or liver (P=0.998).

  • 1

    Seckl JR, Walker BR. 11β-Hydroxysteroid dehydrogenase type 1 – a tissue-specific amplifier of glucocorticoid action. Endocrinology 2001 142 13711376 doi:10.1210/en.142.4.1371.

    • Search Google Scholar
    • Export Citation
  • 2

    Paterson JM, Morton NM, Fievet C, Kenyon CJ, Holmes MC, Staels B, Seckl JR, Mullins JJ. Metabolic syndrome without obesity: hepatic overexpression of 11β-hydroxysteroid dehydrogenase type 1 in transgenic mice. PNAS 2004 101 70887093 doi:10.1073/pnas.0305524101.

    • Search Google Scholar
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  • 3

    Masuzaki H, Paterson J, Shinyama H, Morton NM, Mullins JJ, Seckl JR, Flier JS. A transgenic model of visceral obesity and the metabolic syndrome. Science 2001 294 21662170 doi:10.1126/science.1066285.

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    Walker BR, Connacher AA, Lindsay RM, Webb DJ, Edwards CRW. Carbenoxolone increases hepatic insulin sensitivity in man: a novel role for 11-oxosteroid reductase in enhancing glucocorticoid receptor activation. Journal of Clinical Endocrinology and Metabolism 1995 80 31553159 doi:10.1210/jc.80.11.3155.

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  • 5

    Hughes KA, Webster SP, Walker BR. 11-Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) inhibitors in type 2 diabetes mellitus and obesity. Expert Opinion on Investigational Drugs 2008 17 481496 doi:10.1517/13543784.17.4.481.

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    Alberts P, Engblom L, Edling N, Forsgren M, Klingstrom G, Larsson C, Ronquist-Nii Y, Ohman B, Abrahmsen L. Selective inhibition of 11beta-hydroxysteroid dehydrogenase type 1 decreases blood glucose concentrations in hyperglycaemic mice. Diabetologia 2002 45 15281532 doi:10.1007/s00125-002-0959-6.

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  • 7

    Kotelevtsev YV, Holmes MC, Burchell A, Houston PM, Scholl D, Jamieson PM, Best R, Brown RW, Edwards CRW, Seckl JR, Mullins JJ. 11β-Hydroxysteroid dehydrogenase type 1 knockout mice show attenuated glucocorticoid inducible responses and resist hyperglycaemia on obesity and stress. PNAS 1997 94 1492414929 doi:10.1073/pnas.94.26.14924.

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  • 8

    Morton NM, Holmes MC, Fievet C, Staels B, Tailleux A, Mullins JJ, Seckl JR. Improved lipid and lipoprotein profile, hepatic insulin sensitivity, and glucose tolerance in 11β-hydroxysteroid dehydrogenase type 1 null mice. Journal of Biological Chemistry 2001 276 4129341300 doi:10.1074/jbc.M103676200.

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  • 9

    Morton NM, Paterson JM, Masuzaki H, Holmes MC, Staels B, Fievet C, Walker BR, Flier JS, Mullins JJ, Seckl JR. Novel adipose tissue-mediated resistance to diet-induced visceral obesity in 11β-hydroxysteroid dehydrogenase type 1 deficient mice. Diabetes 2004 3 931938 doi:10.2337/diabetes.53.4.931.

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  • 10

    Rask E, Olsson T, Soderberg S, Andrew R, Livingstone DEW, Johnson O, Walker BR. Tissue-specific dysregulation of cortisol metabolism in human obesity. Journal of Clinical Endocrinology and Metabolism 2001 86 14181421 doi:10.1210/jc.86.3.1418.

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  • 11

    Stewart PM, Boulton A, Kumar S, Clark PMS, Shackleton CHL. Cortisol metabolism in human obesity: impaired cortisone – cortisol conversion in subjects with central adiposity. Journal of Clinical Endocrinology and Metabolism 1999 84 10221027 doi:10.1210/jc.84.3.1022.

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  • 12

    Rodin A, Thakkar H, Taylor N, Clayton R. Hyperandrogenism in polycystic ovary syndrome: evidence of dysregulation of 11beta-hydroxysteroid dehydrogenase. New England Journal of Medicine 1994 330 460465 doi:10.1056/NEJM199402173300703.

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  • 13

    Gambineri A, Vicennati V, Tomassoni F, Pagotto U, Pasquali R, Walker BR. Genetic variation in 11β-hydroxysteroid dehydrogenase type 1 predicts hyperandrogenism amongst lean women with polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 2006 91 22952302 doi:10.1210/jc.2005-2222.

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  • 14

    Draper N, Walker EA, Bujalska IJ, Tomlinson JW, Chalder SM, Arlt W, Lavery G, Bedendo O, Ray DW, Laing I, Malunowicz E, White PC, Hewison M, Mason PJ, Connell JM, Shackleton CHL, Stewart PM. Mutations in the genes encoding 11β-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase interact to cause cortisone reductase deficiency. Nature Genetics 2003 34 434439 doi:10.1038/ng1214.

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  • 15

    Nair S, Lee YH, Lindsay RS, Walker BR, Tataranni PA, Bogardus C, Baier LJ, Permana PA. 11Beta-hydroxysteroid dehydrogenase type 1: genetic polymorphisms are associated with type 2 diabetes in Pima Indians independently of obesity and expression in adipocyte and muscle. Diabetologia 2004 47 10881095 doi:10.1007/s00125-004-1407-6.

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  • 16

    Franks PW, Knowler WC, Nair S, Koska J, Lee Y-H, Lindsay RS, Walker BR, Looker HC, Permana PA, Tataranni PA, Hanson RL. Interaction between an 11beta HSD1 gene variant and birth era modifies the risk of hypertension in Pima Indians. Hypertension 2004 44 681688 doi:10.1161/01.HYP.0000144294.28985.d5.

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  • 17

    Morales MA, Carvajal CA, Ortiz E, Mosso LM, Artigas RA, Owen GI, Fardella CE. Possible pathogenetic role of 11 beta-hydroxysteroid dehydrogenase type 1 (11beta HSD1) gene polymorphisms in arterial hypertension. Revista Médica de Chile 2008 136 701710 doi:10.4067/S0034-98872008000600003.

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  • 18

    Smit P, Dekker MJ, de Jong FJ, Van Den Beld AW, Koper JW, Pols HA, Brinkmann AO, de Jong FH, Breteler MM, Lamberts SW. Lack of association of the 11beta-hydroxysteroid dehydrogenase type 1 gene 83,557insA and hexose-6-phosphate dehydrogenase gene R453Q polymorphisms with body composition, adrenal androgen production, blood pressure, glucose metabolism, and dementia. Journal of Clinical Endocrinology and Metabolism 2007 92 359362 doi:10.1210/jc.2006-1349.

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  • 19

    Draper N, Powell BL, Franks S, Conway GS, Stewart PM, McCarthy MI. Variants implicated in cortisone reductase deficiency do not contribute to susceptibility to common forms of polycystic ovary syndrome. Clinical Endocrinology 2006 65 6470 doi:10.1111/j.1365-2265.2006.02547.x.

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  • 20

    Millan JL, Botella-Carratero JI, Alvarez-Blasco F, Luque-Ramirez M, Sancho J, Moghetti P, Escobar-Morreal HF. A study of the hexose-6-phosphate dehydrogenase gene R453Q and 11beta-hydroxysteroid dehydrogenase type 1 gene 83557insA polymorphisms in the polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 2005 90 41574162 doi:10.1210/jc.2004-1523.

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  • 21

    White PC. Genotypes at 11beta-hydroxysteroid dehydrogenase type 11B1 and hexose-6-phosphate dehydrogenase loci are not risk factors for apparent cortisone reductase deficiency in a large population-based sample. Journal of Clinical Endocrinology and Metabolism 2005 90 58805883 doi:10.1210/jc.2005-0942.

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  • 22

    Andrew R, Smith K, Jones GC, Walker BR. Distinguishing the activities of 11β-hydroxysteroid dehydrogenases in vivo using isotopically labelled cortisol. Journal of Clinical Endocrinology and Metabolism 2002 87 277285 doi:10.1210/jc.87.1.277.

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  • 23

    Gambineri A, Pelusi C, Genghini S, Morselli-Labate AM, Cacciari M, Pagotto U, Pasquali R. Effect of flutamide and metformin administered alone or in combination in dieting obese women with polycystic ovary syndrome. Clinical Endocrinology 2004 60 241249 doi:10.1111/j.1365-2265.2004.01973.x.

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  • 24

    The Rotterdam ESHRE/ASMR-sponsored PCOS consensus workshop group Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome Human Reproduction 2004 19 4147 doi:10.1093/humrep/deh098.

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    Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults Executive summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Journal of the American Medical Association 2001 285 24862497 doi:10.1001/jama.285.19.2486.

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    Gambineri A, Repaci A, Patton L, Grassi I, Pocognoli P, Cognigni GE, Pasqui F, Pagotto U, Pasquali R. Prominent role of low HDL-cholesterol in explaining the high prevalence of the metabolic syndrome in polycystic ovary syndrome. Nutrition, Metabolism, and Cardiovascular Diseases 2009 19 797804 doi:10.1016/j.numecd.2009.01.007.

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  • 27

    Stimson RH, Johnstone AM, Homer NZ, Wake DJ, Morton NM, Andrew R, Lobley GE, Walker BR. Dietary macronutrient content alters cortisol metabolism independently of body weight changes in obese men. Journal of Clinical Endocrinology and Metabolism 2007 92 44804484 doi:10.1210/jc.2007-0692.

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    Hoogendoorn B, Owen MJ, Oefner PJ, Williams N, Austin J, O'Donovan MC. Genotyping single nucleotide polymorphisms by primer extension and high performance liquid chromatography. Human Genetics 1999 104 8993 doi:10.1007/s004390050915.

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  • 29

    Vicennati V, Pasquali R. Abnormalities of the hypothalamic–pituitary–adrenal axis in nondepressed women with abdominal obesity and relations with insulin resistance: evidence for a central and a peripheral alteration. Journal of Clinical Endocrinology and Metabolism 2000 85 40934098 doi:10.1210/jc.85.11.4093.

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    Pasquali R, Gambineri A, Anconetani B, Vicennati V, Colitta D, Caramelli E, Casimirri F, Morselli-Labate AM. The natural history of the metabolic syndrome in young women with the polycystic ovary syndrome and the effect of long-term oestrogen–progestagen treatment. Clinical Endocrinology 1999 50 517527 doi:10.1046/j.1365-2265.1999.00701.x.

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  • 31

    Stimson RH, Andersson J, Andrew R, Redhead DN, Karpe F, Hayes PC, Olsson T, Walker BR. Cortisol release from adipose tissue by 11beta-hydroxysteroid dehydrogenase type 1 in humans. Diabetes 2009 58 4653 doi:10.2337/db08-0969.

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  • 32

    Best R, Walker BR. Additional value of measurement of urinary cortisone and unconjugated cortisol metabolites in assessing the activity of 11β-hydroxysteroid dehydrogenase in vivo. Clinical Endocrinology 1997 47 231236 doi:10.1046/j.1365-2265.1997.2471061.x.

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  • 33

    Malavasi EL, Kelly V, Nath N, Gambineri A, Dakin RS, Pagotto U, Pasquali R, Walker BR, Chapman KE. Functional effects of polymorphisms in the human gene encoding 11beta hydroxysteroid dehydrogenase type 1 (11 beta-HSD1): a sequence variant at the translation start of 11 beta-HSD1 alters enzyme levels. Endocrinology 2010 151 195202 doi:10.1210/en.2009-0663.

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  • 34

    Basu R, Singh RJ, Basu A, Chittilapilly EG, Johnson CM, Toffolo G, Cobelli C, Rizza RA. Splanchnic cortisol production occurs in humans – evidence for conversion of cortisone to cortisol via the 11-β hydroxysteroid dehydrogenase type 1 pathway. Diabetes 2004 53 20512059 doi:10.2337/diabetes.53.8.2051.

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  • 35

    Andrew R, Westerbacka J, Wahren J, Yki-Jarvinen H, Walker BR. The contribution of visceral adipose tissue to splanchnic cortisol production in healthy humans. Diabetes 2005 54 13641370 doi:10.2337/diabetes.54.5.1364.

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  • 36

    Bruley C, Lyons V, Worsley AG, Wilde MD, Darlington GD, Morton NM, Seckl JR, Chapman KE. A novel promoter for the 11beta-hydroxysteroid dehydrogenase type 1 gene is active in lung and is C/EBPalpha independent. Endocrinology 2006 147 28792885 doi:10.1210/en.2005-1621.

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  • 37

    Sai S, Esteves CL, Kelly V, Michailidou Z, Anderson K, Coll AP, Nakagawa Y, Ohzeki T, Seckl JR, Chapman KE. Glucocorticoid regulation of the promoter of 11beta-hydroxysteroid dehydrogenase type 1 is indirect and requires CCAAT/enhancer-binding protein-beta. Molecular Endocrinology 2008 22 20492060 doi:10.1210/me.2007-0489.

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  • 38

    Lavery G, Walker EA, Draper N, Jeyasuria P, Marcos J, Shackleton CHL, Parker KL, White PC, Stewart PM. Hexose-6-phosphate dehydrogenase knock-out mice lack 11β-hydroxysteroid dehydrogenase type 1-mediated glucocorticoid regeneration. Journal of Biological Chemistry 2006 281 65466551 doi:10.1074/jbc.M512635200.

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  • 39

    Lavery GG, Walker EA, Tiganescu A, Ride JP, Shackleton CH, Tomlinson JW, Connell JM, Ray DW, Biason-Lauber A, Malunowicz EM, Arlt W, Stewart PM. Steroid biomarkers and genetic studies reveal inactivating mutations in hexose-6-phosphate dehydrogenase in patients with cortisone reductase deficiency. Journal of Clinical Endocrinology and Metabolism 2008 93 38273832 doi:10.1210/jc.2008-0743.

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  • 40

    Wake DJ, Rask E, Livingstone DEW, Soderberg S, Olsson T, Walker BR. Local and systemic impact of transcriptional upregulation of 11β-hydroxysteroid dehydrogenase type 1 in adipose tissue in human obesity. Journal of Clinical Endocrinology and Metabolism 2003 88 29832988 doi:10.1210/jc.2003-030286.

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