Effects of Obesity and Insulin on Tissue-Specific Recycling Between Cortisol and Cortisone in Men.

CONTEXT: 11ß-Hydroxysteroid dehydrogenase 1 (11ßHSD1) reduces inert cortisone into active cortisol but also catalyzes reverse dehydrogenase activity. Drivers of cortisol/cortisone equilibrium are unclear. With obesity, 11ßHSD1 transcripts are more abundant in adipose, but the consequences for oxidation vs reduction remain unknown. OBJECTIVE: This work aimed to determine whether 11ßHSD1 equilibrium in metabolic tissues is regulated by insulin and obesity. METHODS: A 2-phase, randomized, crossover, single-blinded study in a clinical research facility was conducted of 10 lean and obese healthy men. 11ß-Reductase and 11ß-dehydrogenase activities were measured during infusion of 9,11,12,12-[2H]4-cortisol and 1,2-[2H]2-cortisone, respectively, on 2 occasions: once during saline infusion and once during a hyperinsulinemic-euglycemic clamp. Arterialized and venous samples were obtained across forearm skeletal muscle and abdominal subcutaneous adipose. Steroids were quantified by liquid chromatography-tandem mass spectrometry and adipose tissue transcripts by quantitative polymerase chain reaction. RESULTS: Neither whole-body nor tissue-specific rates of production of cortisol or cortisone differed between lean and obese men, however insulin attenuated the diurnal decrease. Whole-body 11ß-HSD1 reductase activity tended to be higher in obesity (~ 10%) and was further increased by insulin. Across adipose tissue, 11ß-reductase activity was detected in obese individuals only and increased in the presence of insulin (18.99 ±â€…9.62 vs placebo 11.68 ±â€…3.63 pmol/100 g/minute; P < .05). Across skeletal muscle, 11ß-dehydrogenase activity was reduced by insulin in lean men only (2.55 ±â€…0.90 vs 4.50 ±â€…1.42 pmol/100 g/minute, P < .05). CONCLUSIONS: Regeneration of cortisol is upregulated by insulin in adipose tissue but not skeletal muscle. In obesity, the equilibrium between 11ß-reductase and 11ß-dehydrogenase activities likely promotes cortisol accumulation in adipose, which may lead to adverse metabolic consequences.

'CPR for Feet' care bundle to improve foot assessment in inpatient diabetes.

AIMS: The Scottish Inpatient Diabetes Foot Audit conducted in 2013 revealed that 57% of inpatients had not had their feet checked on admission, 60% of those at risk did not have pressure relief in place and 2.4% developed a new foot lesion. In response, the Scottish Diabetes Foot Action Group launched the 'CPR for Feet' campaign. The aim of this project was to raise awareness of the 'Check, Protect and Refer' (CPR) campaign as well as improve the assessment and management of inpatients with diabetes. METHODS: A quality improvement project underpinned by Plan-Do-Study-Act (PDSA) methodology was undertaken. The first and second cycles focused on staff education and the implementation of a 'CPR for Feet' assessment checklist using campaign guidelines, training manuals and modules. The third and fourth cycles focused on staff feedback and the implementation of a 'CPR for Feet' care bundle. RESULTS: Baseline measurements revealed 28% of patients had evidence of foot assessment. Medical and nursing staff reported to be largely unaware of the 'CPR for Feet' campaign (13%). Fifty-two per cent of inpatients with diabetes had their feet assessed and managed correctly following the second PDSA cycle. After completion of the third and fourth PDSA this number improved further to 72% and all staff reported to be aware of the campaign. CONCLUSIONS: The introduction of a 'CPR for Feet' care bundle improved the assessment of inpatients with diabetes.

Simultaneous pharmacokinetic and pharmacodynamic analysis of 5α-reductase inhibitors and androgens by liquid chromatography tandem mass spectrometry.

Benign prostatic hyperplasia and prostate cancer can be treated with the 5α-reductase inhibitors, finasteride and dutasteride, when pharmacodynamic biomarkers are useful in assessing response. A novel method was developed to measure the substrates and products of 5α-reductases (testosterone, 5α-dihydrotestosterone (DHT), androstenedione) and finasteride and dutasteride simultaneously by liquid chromatography tandem mass spectrometry, using an ABSciex QTRAP(®) 5500, with a Waters Acquity™ UPLC. Analytes were extracted from serum (500 µL) via solid-phase extraction (Oasis(®) HLB), with (13)C3-labelled androgens and d9-finasteride included as internal standards. Analytes were separated on a Kinetex C18 column (150 × 3 mm, 2.6 µm), using a gradient run of 19 min. Temporal resolution of analytes from naturally occurring isomers and mass +2 isotopomers was ensured. Protonated molecular ions were detected in atmospheric pressure chemical ionisation mode and source conditions optimised for DHT, the least abundant analyte. Multiple reaction monitoring was performed as follows: testosterone (m/z 289 → 97), DHT (m/z 291 → 255), androstenedione (m/z 287 → 97), dutasteride (m/z 529 → 461), finasteride (m/z 373 → 317). Validation parameters (intra- and inter-assay precision and accuracy, linearity, limits of quantitation) were within acceptable ranges and biological extracts were stable for 28 days. Finally the method was employed in men treated with finasteride or dutasteride; levels of DHT were lowered by both drugs and furthermore the substrate concentrations increased.

Stress hyperglycaemia in hospitalised patients and their 3-year risk of diabetes: a Scottish retrospective cohort study.

BACKGROUND: Hyperglycaemia during hospital admission is common in patients who are not known to have diabetes and is associated with adverse outcomes. The risk of subsequently developing type 2 diabetes, however, is not known. We linked a national database of hospital admissions with a national register of diabetes to describe the association between admission glucose and the risk of subsequently developing type 2 diabetes. METHODS AND FINDINGS: In a retrospective cohort study, patients aged 30 years or older with an emergency admission to hospital between 2004 and 2008 were included. Prevalent and incident diabetes were identified through the Scottish Care Information (SCI)-Diabetes Collaboration national registry. Patients diagnosed prior to or up to 30 days after hospitalisation were defined as prevalent diabetes and were excluded. The predicted risk of developing incident type 2 diabetes during the 3 years following hospital discharge by admission glucose, age, and sex was obtained from logistic regression models. We performed separate analyses for patients aged 40 and older, and patients aged 30 to 39 years. Glucose was measured in 86,634 (71.0%) patients aged 40 and older on admission to hospital. The 3-year risk of developing type 2 diabetes was 2.3% (1,952/86,512) overall, was <1% for a glucose ≤ 5 mmol/l, and increased to approximately 15% at 15 mmol/l. The risks at 7 mmol/l and 11.1 mmol/l were 2.6% (95% CI 2.5-2.7) and 9.9% (95% CI 9.2-10.6), respectively, with one in four (21,828/86,512) and one in 40 (1,798/86,512) patients having glucose levels above each of these cut-points. For patients aged 30-39, the risks at 7 mmol/l and 11.1 mmol/l were 1.0% (95% CI 0.8-1.3) and 7.8% (95% CI 5.7-10.7), respectively, with one in eight (1,588/11,875) and one in 100 (120/11,875) having glucose levels above each of these cut-points. The risk of diabetes was also associated with age, sex, and socio-economic deprivation, but not with specialty (medical versus surgical), raised white cell count, or co-morbidity. Similar results were obtained for pre-specified sub-groups admitted with myocardial infarction, chronic obstructive pulmonary disease, and stroke. There were 25,193 deaths (85.8 per 1,000 person-years) over 297,122 person-years, of which 2,406 (8.1 per 1,000 person-years) were attributed to vascular disease. Patients with glucose levels of 11.1 to 15 mmol/l and >15 mmol/l had higher mortality than patients with a glucose of <6.1 mmol/l (hazard ratio 1.54; 95% CI 1.42-1.68 and 2.50; 95% CI 2.14-2.95, respectively) in models adjusting for age and sex. Limitations of our study include that we did not have data on ethnicity or body mass index, which may have improved prediction and the results have not been validated in non-white populations or populations outside of Scotland. CONCLUSION: Plasma glucose measured during an emergency hospital admission predicts subsequent risk of developing type 2 diabetes. Mortality was also 1.5-fold higher in patients with elevated glucose levels. Our findings can be used to inform patients of their long-term risk of type 2 diabetes, and to target lifestyle advice to those patients at highest risk. Please see later in the article for the Editors' Summary.

5α-reductase type 1 modulates insulin sensitivity in men.

CONTEXT: 5α-Reductase (5αR) types 1 and 2 catalyze the A-ring reduction of steroids, including androgens and glucocorticoids. 5α-R inhibitors lower dihydrotestosterone in benign prostatic hyperplasia; finasteride inhibits 5αR2, and dutasteride inhibits both 5αR2 and 5αR1. In rodents, loss of 5αR1 promotes fatty liver. OBJECTIVE: Our objective was to test the hypothesis that inhibition of 5αR1 causes metabolic dysfunction in humans. DESIGN, SETTING, AND PARTICIPANTS: This double-blind randomized controlled parallel group study at a clinical research facility included 46 men (20-85 years) studied before and after intervention. INTERVENTION: Oral dutasteride (0.5 mg daily; n = 16), finasteride (5 mg daily; n = 16), or control (tamsulosin; 0.4 mg daily; n = 14) was administered for 3 months. MAIN OUTCOME MEASURE: Glucose disposal was measured during a stepwise hyperinsulinemic-euglycemic clamp. Data are mean (SEM). RESULTS: Dutasteride and finasteride had similar effects on steroid profiles, with reduced urinary androgen and glucocorticoid metabolites and reduced circulating DHT but no change in plasma or salivary cortisol. Dutasteride, but not finasteride, reduced stimulation of glucose disposal by high-dose insulin (dutasteride by -5.7 [3.2] µmol/kg fat-free mass/min, versus finasteride +7.2 [3.0], and tamsulosin +7.0 [2.0]). Dutasteride also reduced suppression of nonesterified fatty acids by insulin and increased body fat (by 1.6% [0.6%]). Glucose production and glycerol turnover were unchanged. Consistent with metabolic effects of dutasteride being mediated in peripheral tissues, mRNA for 5αR1 but not 5αR2 was detected in human adipose tissue. CONCLUSION: Dual inhibition of 5αRs, but not inhibition of 5αR2 alone, modulates insulin sensitivity in human peripheral tissues rather than liver. This may have important implications for patients prescribed dutasteride for prostatic disease.

Relative adrenal insufficiency in mice deficient in 5α-reductase 1.

Patients with critical illness or hepatic failure exhibit impaired cortisol responses to ACTH, a phenomenon known as 'relative adrenal insufficiency'. A putative mechanism is that elevated bile acids inhibit inactivation of cortisol in liver by 5α-reductases type 1 and type 2 and 5ß-reductase, resulting in compensatory downregulation of the hypothalamic-pituitary-adrenal axis and adrenocortical atrophy. To test the hypothesis that impaired glucocorticoid clearance can cause relative adrenal insufficiency, we investigated the consequences of 5α-reductase type 1 deficiency in mice. In adrenalectomised male mice with targeted disruption of 5α-reductase type 1, clearance of corticosterone was lower after acute or chronic (eightfold, P<0.05) administration, compared with WT control mice. In intact 5α-reductase-deficient male mice, although resting plasma corticosterone levels were maintained, corticosterone responses were impaired after ACTH administration (26% lower, P<0.05), handling stress (2.5-fold lower, P<0.05) and restraint stress (43% lower, P<0.05) compared with WT mice. mRNA levels of Nr3c1 (glucocorticoid receptor), Crh and Avp in pituitary or hypothalamus were altered, consistent with enhanced negative feedback. These findings confirm that impaired peripheral clearance of glucocorticoids can cause 'relative adrenal insufficiency' in mice, an observation with important implications for patients with critical illness or hepatic failure, and for patients receiving 5α-reductase inhibitors for prostatic disease.

Displacement of cortisol from human heart by acute administration of a mineralocorticoid receptor antagonist.

CONTEXT: Mineralocorticoid receptor (MR) antagonists have beneficial effects in patients with heart failure and myocardial infarction, often attributed to blocking aldosterone action in the myocardium. However, binding of aldosterone to MR requires local activity of the enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), which inactivates cortisol to cortisone and thereby prevents receptor occupancy by cortisol. In vivo activity of 11ß-HSD2 and potential occupancy of MR by cortisol in human heart have not been quantified. OBJECTIVE: This study aimed to measure in vivo activity of 11ß-HSD2 and to establish whether cortisol binds MR in human heart. PARTICIPANTS AND INTERVENTIONS: Nine patients without heart failure undergoing diagnostic coronary angiography were infused to steady state with the stable isotope tracers 9,11,12,12-[(2)H]4-cortisol and 1,2-[(2)H]2-cortisone to quantify cortisol and cortisone production. Samples were obtained from the femoral artery and coronary sinus before and for 40 minutes after bolus iv administration of an MR antagonist, potassium canrenoate. Coronary sinus blood flow was measured by venography and Doppler flow wire. RESULTS: There was no detectable production of cortisol or cortisone across the myocardium. After potassium canrenoate administration, plasma aldosterone concentrations increased substantially but aldosterone was not detectably released from the myocardium. In contrast, plasma cortisol concentrations did not change in the systemic circulation but tissue-bound cortisol was released transiently from the myocardium after potassium canrenoate administration. CONCLUSIONS: Human cardiac 11ß-HSD2 activity appears too low to inactivate cortisol to cortisone. Cortisol is displaced acutely from the myocardium by MR antagonists and may contribute to adverse MR activation in human heart.

Measurement of tamsulosin in human serum by liquid chromatography-tandem mass spectrometry.

A simple, sensitive and robust method to extract tamsulosin from human serum, and quantify by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated and is applicable as a measure of compliance in clinical research. Tamsulosin was extracted from human serum (100µL) via liquid-liquid extraction with methyl tert-butyl ether (2mL) following dilution with 0.1M ammonium hydroxide (100µL), achieving 99.9% analyte recovery. Internal standard, d9-finasteride, was synthesised in-house. Analyte and internal standard were separated on an Ascentis(®) Express C18 (100mm×3mm, 2.7µm) column using a gradient elution with mobile phases methanol and 2mM aqueous ammonium acetate (5:95, v/v). Total run-time was 6min. Tamsulosin was quantified using a triple quadrupole mass spectrometer operated in multi-reaction-monitoring (MRM) mode using positive electrospray ionisation. Mass transitions monitored for quantitation were: tamsulosin m/z 409→228 and d9-finasteride m/z 382→318, with the structural formulae of ions confirmed by Fourier transform ion cyclotron resonance mass spectrometry (within 10ppm). The limit of quantitation was 0.2ng/mL, and the method was validated in the linear range 0.2-50ng/mL with acceptable inter- and intra-assay precision and accuracy and stability suitable for routine laboratory practice. The method was successfully applied to samples taken from research volunteers in a clinical study of benign prostatic hyperplasia.

Recycling between cortisol and cortisone in human splanchnic, subcutaneous adipose, and skeletal muscle tissues in vivo.

11ß-Hydroxysteroid dehydrogenase type 1 (11ßHSD1) is a therapeutic target in metabolic syndrome because it catalyses reductase regeneration of cortisol from cortisone in adipose and liver. 11ßHSD1 can also catalyze the reverse dehydrogenase reaction in vitro (e.g., if cofactor is limited). We used stable isotope tracers to test the hypothesis that both 11ßHSD1-reductase and -dehydrogenase activities occur in human metabolic tissues in vivo. 1,2-[(2)H](2)-Cortisone (d2-cortisone) was validated as a tracer for 11ß-dehydrogenase activity and its inhibition by licorice. d2-Cortisone and 9,11,12,12-[(2)H](4)-cortisol (d4-cortisol) (to measure 11ß-reductase activity) were coinfused and venous samples obtained from skeletal muscle, subcutaneous adipose (n = 6), and liver (n = 4). Steroids were measured by liquid chromatography-tandem mass spectrometry and arteriovenous differences adjusted for blood flow. Data are means ± SEM. 11ß-Reductase and -dehydrogenase activities were detected in muscle (cortisol release 19.7 ± 4.1 pmol/100 mL/min, d3-cortisol 5.9 ± 1.8 pmol/100 mL/min, and cortisone 15.2 ± 5.8 pmol/100 mL/min) and splanchnic (cortisol 64.0 ± 11.4 nmol/min, d3-cortisol 12.9 ± 2.1 nmol/min, and cortisone 19.5 ± 2.8 nmol/min) circulations. In adipose, dehydrogenase was more readily detected than reductase (cortisone release 38.7 ± 5.8 pmol/100 g/min). Active recycling between cortisol and cortisone in metabolic tissues in vivo may facilitate dynamic control of intracellular cortisol but makes consequences of dysregulation of 11ßHSD1 transcription in obesity and diabetes unpredictable. Disappointing efficacy of 11ßHSD1 inhibitors in phase II studies could be explained by lack of selectivity for 11ß-reductase.

Glucocorticoids turn over slowly in human adipose tissue in vivo.

CONTEXT: Lipophilic plasma glucocorticoids are thought to gain rapid access to intracellular compartments in adipose tissue. In other organs, transport can be regulated in a steroid- and tissue-specific manner. Moreover, 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) generates additional cortisol within adipose. AIM: The aim was to measure the rate of exchange of cortisol between plasma and adipose for comparison with rates of intracellular cortisol generation by 11ßHSD1. PARTICIPANTS AND INTERVENTIONS: With ethical approval, otherwise healthy females (n = 6) undergoing hysterectomy for benign indications were infused with tracer 9,11,12,12-[(2)H](4)cortisol (d4-cortisol). Adipose biopsies and peripheral venous samples were obtained during surgery after 3.9-5.5 h of infusion. Glucocorticoids were quantified using liquid chromatography tandem mass spectrometry. RESULTS: In plasma, d4-cortisol concentrations and appearance rates of cortisol and d3-cortisol (reflecting 11ßHSD1 activity) did not change during surgery. In both omental and sc adipose, cortisol concentrations were lower than in plasma, consistent with differences in corticosteroid binding globulin, and enrichment with d4-cortisol was low (sc, 7.2 ± 0.6%; omental, 7.4 ± 0.7%; vs. plasma, 15.5 ± 1.0%). The rate of accumulation of d4-cortisol in adipose depots was 0.5 ± 0.1 (sc) and 0.4 ± 0.1 (omental) nmol/kg · h, and the proportion of intraadipose cortisol replaced each hour only 10.7 ± 1.0 and 10.4 ± 0.7%, respectively. The contribution of 11ßHSD1 to this turnover could not be quantified because very little substrate d3-cortisone accumulated in adipose during infusion. CONCLUSIONS: Slow turnover of the adipose glucocorticoid pool suggests that rapid acute fluctuations in circulating cortisol are not reflected in adipose, so that 11ßHSD1 activity (previously estimated to generate 9 nmol cortisol/kg · h in sc adipose) may play a relatively important role in modulating activation of glucocorticoid receptors.

11-Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) inhibitors in type 2 diabetes mellitus and obesity.

BACKGROUND: Glucocorticoids such as cortisol are important regulators of fuel metabolism during starvation and stress. Chronic glucocorticoid excess induces obesity with multiple features of the metabolic syndrome. OBJECTIVE: In this article, we review the importance of glucocorticoids in metabolic syndrome and the approaches that have been explored to reduce glucocorticoid action as the basis for novel therapy of Type 2 diabetes and obesity. METHOD: We focus on the enzyme 11-beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which amplifies glucocorticoid concentrations in key metabolic tissues including liver and adipose tissue. RESULTS/CONCLUSION: Several 11beta-HSD1 inhibitors are in late preclinical or early clinical development and we review here the properties of the class leaders and their potential as the next generation of drugs with multiple benefits in metabolic syndrome.