Altered cortisol metabolism in individuals with HNF1A-MODY.

OBJECTIVE AND CONTEXT: Maturity onset diabetes of the young due to variants in HNF1A (HNF1A-MODY) is the most common form of monogenic diabetes. Individuals with HNF1A-MODY usually have a lean phenotype which contrasts with type 2 diabetes (T2DM). Data from hepatocytes derived from Hnf1a knock-out mice demonstrated dysregulation of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which regulates glucocorticoid availability and action in target tissues, together with 11ß-HSD2 and steroid A-ring reductases, 5α- and 5ß-reductase. We proposed that altered glucocorticoid metabolism might underpin some of the phenotypic differences between patients with HNF1A-MODY and those with T2DM. DESIGN: A retrospective matched cohort study. PATIENTS AND MEASUREMENTS: 24-hours urine steroid metabolome profiling was carried out by gas chromatography-mass spectrometry in 35 subjects with HNF1A-MODY, 35 individuals with T2DM and 35 healthy controls matched for age, sex and BMI. The steroid metabolites were expressed as µg/L in all groups and measured in mid-morning urine in diabetic subjects and 24-hour urine collection in healthy controls. Hence, only ratios were compared not the individual steroids. Established ratios of glucocorticoid metabolites were used to estimate 11ß-HSD1/2 and 5α- and 5ß-reductase activities. RESULTS: While 11ß-HSD1 activity was similar in all groups, 11ß-HSD2 activity was significantly lower in subjects with HNF1A-MODY and T2DM than in healthy controls. The ratio of 5ß- to 5α-metabolites of cortisol was higher in subjects with HNF1A-MODY than in T2DM and healthy controls, probably due to increased activity of the 5ß-reductase (AKR1D1) in HNF1A-MODY. CONCLUSIONS: This is the first report of steroid metabolites in HNF1A-MODY. We have identified distinct differences in steroid metabolism pathways in subjects with HNF1A-MODY that have the potential to alter steroid hormone availability. Further studies are required to explore whether these changes link to phenotype.

Increased central adiposity and decreased subcutaneous adipose tissue 11ß-hydroxysteroid dehydrogenase type 1 are associated with deterioration in glucose tolerance-A longitudinal cohort study.

OBJECTIVE AND CONTEXT: Increasing adiposity, ageing and tissue-specific regeneration of cortisol through the activity of 11ß-hydroxysteroid dehydrogenase type 1 have been associated with deterioration in glucose tolerance. We undertook a longitudinal, prospective clinical study to determine if alterations in local glucocorticoid metabolism track with changes in glucose tolerance. DESIGN, PATIENTS, AND MEASUREMENTS: Sixty-five overweight/obese individuals (mean age 50.3 ± 7.3 years) underwent oral glucose tolerance testing, body composition assessment, subcutaneous adipose tissue biopsy and urinary steroid metabolite analysis annually for up to 5 years. Participants were categorized into those in whom glucose tolerance deteriorated ("deteriorators") or improved ("improvers"). RESULTS: Deteriorating glucose tolerance was associated with increasing total and trunk fat mass and increased subcutaneous adipose tissue expression of lipogenic genes. Subcutaneous adipose tissue 11ß-HSD1 gene expression decreased in deteriorators, and at study completion, it was highest in the improvers. There was a significant negative correlation between change in area under the curve glucose and 11ß-HSD1 expression. Global 11ß-HSD1 activity did not change and was not different between deteriorators and improvers at baseline or follow-up. CONCLUSION: Longitudinal deterioration in metabolic phenotype is not associated with increased 11ß-HSD1 activity, but decreased subcutaneous adipose tissue gene expression. These changes may represent a compensatory mechanism to decrease local glucocorticoid exposure in the face of an adverse metabolic phenotype.

TNFα regulates cortisol metabolism in vivo in patients with inflammatory arthritis.

OBJECTIVE: To determine the relationship between inflammation and glucocorticoid metabolism in vivo, in a clinical study of patients with inflammatory arthritis treated with anti-TNFα therapy. METHODS: Urine samples were collected from patients with rheumatoid arthritis (RA) and psoriatic arthritis (PsA) as part of a multicentre study assessing responses to infliximab and etanercept. Systemic measures of glucocorticoid metabolism were assessed by gas chromatography/mass spectrometry at weeks 0 (baseline), 4 and 12 after anti-TNFα therapy. Clinical data including DAS28 and C-reactive protein were also collected. RESULTS: Systemic measures of 11ß-HSD1 activity in patients with inflammatory arthritis decreased significantly following anti-TNFα therapy in patients with RA and PsA. Additionally, the activity of the glucocorticoid inactivating enzyme 5α-reductase appeared to increase significantly. CONCLUSIONS: This study demonstrates, for the first time, that the increased 11ß-HSD1 activity seen in patients with inflammatory arthritis is mediated through TNFα. Furthermore, the changes in related glucocorticoid metabolising enzymes suggest that there is a coordinated change in glucocorticoid metabolism which promotes higher tissue glucocorticoid levels.

Cortisone-reductase deficiency associated with heterozygous mutations in 11beta-hydroxysteroid dehydrogenase type 1.

In peripheral target tissues, levels of active glucocorticoid hormones are controlled by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), a dimeric enzyme that catalyzes the reduction of cortisone to cortisol within the endoplasmic reticulum. Loss of this activity results in a disorder termed cortisone reductase deficiency (CRD), typified by increased cortisol clearance and androgen excess. To date, only mutations in H6PD, which encodes an enzyme supplying cofactor for the reaction, have been identified as the cause of disease. Here we examined the HSD11B1 gene in two cases presenting with biochemical features indicative of a milder form of CRD in whom the H6PD gene was normal. Novel heterozygous mutations (R137C or K187N) were found in the coding sequence of HSD11B1. The R137C mutation disrupts salt bridges at the subunit interface of the 11ß-HSD1 dimer, whereas K187N affects a key active site residue. On expression of the mutants in bacterial and mammalian cells, activity was either abolished (K187N) or greatly reduced (R137C). Expression of either mutant in a bacterial system greatly reduced the yield of soluble protein, suggesting that both mutations interfere with subunit folding or dimer assembly. Simultaneous expression of mutant and WT 11ß-HSD1 in bacterial or mammalian cells, to simulate the heterozygous condition, indicated a marked suppressive effect of the mutants on both the yield and activity of 11ß-HSD1 dimers. Thus, these heterozygous mutations in the HSD11B1 gene have a dominant negative effect on the formation of functional dimers and explain the genetic cause of CRD in these patients.

The effect of spironolactone upon corticosteroid hormone metabolism in patients with early stage chronic kidney disease.

CONTEXT: Aldosterone has emerged as an important mediator of disease progression and mortality in patients with chronic heart and kidney disease (CKD). Despite the increasing use of mineralocorticoid receptor antagonists in these patients, little is known about the effects on corticosteroid hormone secretion and metabolism. OBJECTIVE: To assess corticosteroid hormone secretion and metabolism in patients with early stage CKD before and after spironolactone (Spiro). DESIGN: Randomized, double-blind, placebo-controlled interventional study. SETTING: Single tertiary referral centre. PATIENTS: A total of 112 patients with stable stage 2/3 CKD. INTERVENTIONS: Patients were randomized to receive either Spiro 25 mg once daily or placebo for 36 weeks. MAIN OUTCOME MEASURES: Plasma renin activity (PRA), angiotensin II (AngII) and steroid hormones were analysed by standard assays; urinary corticosteroid hormone metabolites (5α+5ß-tetrahydro-cortisol (5α+5ß-THF), TH-cortisone (THE), 3α5ß-TH-aldosterone (TH-Aldo), 5α+5ß-TH-deoxycorticosterone (5α+5ß-TH-DOC), TH-11-desoxycortisol (THS)) were analysed by gas chromatography/mass spectrometry. RESULTS: Plasma aldosterone concentration (PAC) was inversely correlated with eGFR (r = -0·331, P < 0·001). Urinary 24-h excretion of TH-Aldo was correlated with PAC (r = 0·214, P < 0·05) and diastolic blood pressure (BP) (r = 0·212, P = <0·05), whereas total 24-h urinary cortisol metabolite excretion was correlated with systolic BP (r = 0·316, P < 0·01). In addition, 11ß-hydroxysteroid dehydrogenase (11ß-HSD) type 1 activity (urinary 5α+5ß-THF)/THE) ratio) was correlated with PRA (r = 0·277, P < 0·01). Spiro treatment significantly reduced BP (123 ± 11/76 ± 7 vs 119 ± 11/73 ± 8 mmHg, P < 0·01) despite renin-angiotensin-aldosterone system induction, reflected by increased urinary 24-h TH-Aldo excretion (17·6 (12, 86) vs 26 (18, 80) µg/24 h, P < 0·05). By contrast, Spiro had no effect on total urinary cortisol metabolite excretion, 11ß-hydroxylase, 11ß-HSD type 1 and 2 activity. CONCLUSIONS: Aldo and cortisol are positively associated with BP suggesting that adrenal hyperactivity may in part explain the increased cardiovascular risk in patients with early end-stage CKD. Addition of Spiro had no effect on glucocorticoid metabolism or total 24-h corticosteroid production.

Glucocorticoids regulate AKR1D1 activity in human liver in vitro and in vivo.

Steroid 5ß-reductase (AKR1D1) is highly expressed in human liver where it inactivates endogenous glucocorticoids and catalyses an important step in bile acid synthesis. Endogenous and synthetic glucocorticoids are potent regulators of metabolic phenotype and play a crucial role in hepatic glucose metabolism. However, the potential of synthetic glucocorticoids to be metabolised by AKR1D1 as well as to regulate its expression and activity has not been investigated. The impact of glucocorticoids on AKR1D1 activity was assessed in human liver HepG2 and Huh7 cells; AKR1D1 expression was assessed by qPCR and Western blotting. Genetic manipulation of AKR1D1 expression was conducted in HepG2 and Huh7 cells and metabolic assessments were made using qPCR. Urinary steroid metabolite profiling in healthy volunteers was performed pre- and post-dexamethasone treatment, using gas chromatography-mass spectrometry. AKR1D1 metabolised endogenous cortisol, but cleared prednisolone and dexamethasone less efficiently. In vitro and in vivo, dexamethasone decreased AKR1D1 expression and activity, further limiting glucocorticoid clearance and augmenting action. Dexamethasone enhanced gluconeogenic and glycogen synthesis gene expression in liver cell models and these changes were mirrored by genetic knockdown of AKR1D1 expression. The effects of AKR1D1 knockdown were mediated through multiple nuclear hormone receptors, including the glucocorticoid, pregnane X and farnesoid X receptors. Glucocorticoids down-regulate AKR1D1 expression and activity and thereby reduce glucocorticoid clearance. In addition, AKR1D1 down-regulation alters the activation of multiple nuclear hormone receptors to drive changes in gluconeogenic and glycogen synthesis gene expression profiles, which may exacerbate the adverse impact of exogenous glucocorticoids.

Accurate non-invasive diagnosis and staging of non-alcoholic fatty liver disease using the urinary steroid metabolome.

BACKGROUND: The development of accurate, non-invasive markers to diagnose and stage non-alcoholic fatty liver disease (NAFLD) is critical to reduce the need for an invasive liver biopsy and to identify patients who are at the highest risk of hepatic and cardio-metabolic complications. Disruption of steroid hormone metabolic pathways has been described in patients with NAFLD. AIM(S): To assess the hypothesis that assessment of the urinary steroid metabolome may provide a novel, non-invasive biomarker strategy to stage NAFLD. METHODS: We analysed the urinary steroid metabolome in 275 subjects (121 with biopsy-proven NAFLD, 48 with alcohol-related cirrhosis and 106 controls), using gas chromatography-mass spectrometry (GC-MS) coupled with machine learning-based Generalised Matrix Learning Vector Quantisation (GMLVQ) analysis. RESULTS: Generalised Matrix Learning Vector Quantisation analysis achieved excellent separation of early (F0-F2) from advanced (F3-F4) fibrosis (AUC receiver operating characteristics [ROC]: 0.92 [0.91-0.94]). Furthermore, there was near perfect separation of controls from patients with advanced fibrotic NAFLD (AUC ROC = 0.99 [0.98-0.99]) and from those with NAFLD cirrhosis (AUC ROC = 1.0 [1.0-1.0]). This approach was also able to distinguish patients with NAFLD cirrhosis from those with alcohol-related cirrhosis (AUC ROC = 0.83 [0.81-0.85]). CONCLUSIONS: Unbiased GMLVQ analysis of the urinary steroid metabolome offers excellent potential as a non-invasive biomarker approach to stage NAFLD fibrosis as well as to screen for NAFLD. A highly sensitive and specific urinary biomarker is likely to have clinical utility both in secondary care and in the broader general population within primary care and could significantly decrease the need for liver biopsy.

Hepatic 11beta-HSD1 mRNA expression in fatty liver and nonalcoholic steatohepatitis.

CONTEXT: Nonalcoholic fatty liver disease represents the hepatic manifestation of the metabolic syndrome. Nonalcoholic steatohepatitis (NASH) is the progressive form of liver injury. The pathophysiology that leads to NASH is not well understood. OBJECTIVE: We hypothesize that an altered cortisol metabolism in the liver may be a pathogenetic factor. DESIGN AND PATIENTS: 75 patients (28 men, 47 women) underwent liver biopsy for elevation in liver enzymes. Histological diagnosis identified normal liver in eight, fatty liver in 20, NASH grade 1 in 22, grade 2 in nine, grade 3 in three patients, and other forms of hepatitis or cirrhosis in 13 patients. We quantified hepatic 11beta-hydroxysteroid dehydrogenase type1 (11beta-HSD1) and hexose-6-phosphate-dehydrogenase (H6PDH) mRNA expression by real-time PCR. In addition, analysis of 24 h urinary excretion of cortisol metabolites using GCMS was performed and compared with healthy controls. RESULTS: 11beta-HSD1 mRNA expression correlated significantly (R2= 0.809; P < 0.001) with H6PDH mRNA expression, negatively with waist-to-hip ratio in women (R2= 0.394; P= 0.005), but not with urinary (THF + 5alpha-THF)/THE ratio, total cortisol metabolite excretion, age, BMI, degree of fatty liver or NASH stages. Total cortisol metabolite excretion was increased in patients with fatty liver or NASH compared with healthy controls. CONCLUSIONS: Our data suggest that expression of hepatic 11beta-HSD1 and H6PDH are closely interlinked. 11beta-HSD1 gene expression does not seem to be involved in the pathogenesis of fatty liver or NASH. However, those patients showed an increased 5alpha- and 5beta-reduction of cortisol leading to an increased cortisol turnover rate and an activation of the HPA axis.

AKR1D1 regulates glucocorticoid availability and glucocorticoid receptor activation in human hepatoma cells.

Steroid hormones, including glucocorticoids and androgens, have potent actions to regulate many cellular processes within the liver. The steroid A-ring reductase, 5ß-reductase (AKR1D1), is predominantly expressed in the liver, where it inactivates steroid hormones and, in addition, plays a crucial role in bile acid synthesis. However, the precise functional role of AKR1D1 to regulate steroid hormone action in vitro has not been demonstrated. We have therefore hypothesised that genetic manipulation of AKR1D1 has the potential to regulate glucocorticoid availability and action in human hepatocytes. In both liver (HepG2) and non-liver cell (HEK293) lines, AKR1D1 over-expression increased glucocorticoid clearance with a concomitant decrease in the activation of the glucocorticoid receptor and the down-stream expression of glucocorticoid target genes. Conversely, knockdown of AKR1D1 using siRNA decreased glucocorticoid clearance and reduced the generation of 5ß-reduced metabolites. In addition, the two 5α-reductase inhibitors finasteride and dutasteride failed to effectively inhibit AKR1D1 activity in either cell-free or hepatocellular systems. Through manipulation of AKR1D1 expression and activity, we have demonstrated its potent ability to regulate glucocorticoid availability and receptor activation within human hepatoma cells. These data suggest that AKR1D1 may have an important role in regulating endogenous (and potentially exogenous) glucocorticoid action that may be of particular relevance to physiological and pathophysiological processes affecting the liver.

AKR1D1 is a novel regulator of metabolic phenotype in human hepatocytes and is dysregulated in non-alcoholic fatty liver disease.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome. Steroid hormones and bile acids are potent regulators of hepatic carbohydrate and lipid metabolism. Steroid 5ß-reductase (AKR1D1) is highly expressed in human liver where it inactivates steroid hormones and catalyzes a fundamental step in bile acid synthesis. METHODS: Human liver biopsies were obtained from 34 obese patients and AKR1D1 mRNA expression levels were measured using qPCR. Genetic manipulation of AKR1D1 was performed in human HepG2 and Huh7 liver cell lines. Metabolic assessments were made using transcriptome analysis, western blotting, mass spectrometry, clinical biochemistry, and enzyme immunoassays. RESULTS: In human liver biopsies, AKR1D1 expression decreased with advancing steatosis, fibrosis and inflammation. Expression was decreased in patients with type 2 diabetes. In human liver cell lines, AKR1D1 knockdown decreased primary bile acid biosynthesis and steroid hormone clearance. RNA-sequencing identified disruption of key metabolic pathways, including insulin action and fatty acid metabolism. AKR1D1 knockdown increased hepatocyte triglyceride accumulation, insulin sensitivity, and glycogen synthesis, through increased de novo lipogenesis and decreased ß-oxidation, fueling hepatocyte inflammation. Pharmacological manipulation of bile acid receptor activation prevented the induction of lipogenic and carbohydrate genes, suggesting that the observed metabolic phenotype is driven through bile acid rather than steroid hormone availability. CONCLUSIONS: Genetic manipulation of AKR1D1 regulates the metabolic phenotype of human hepatoma cell lines, driving steatosis and inflammation. Taken together, the observation that AKR1D1 mRNA is down-regulated with advancing NAFLD suggests that it may have a crucial role in the pathogenesis and progression of the disease.

Human Delta4-3-oxosteroid 5beta-reductase (AKR1D1) deficiency and steroid metabolism.

Conclusive in vivo evidence regarding the enzyme responsible for steroid hormone 5beta-reduction has not been obtained, although studies have suggested it may be the same enzyme as that utilized for cholic acid and chenodeoxycholic bile-acid synthesis. We have recorded the steroid metabolome of a patient with a defect in the "bile-acid" 5beta-reductase (AKR1D1) and from this confirm that this enzyme is additionally responsible for steroid hormone metabolism. The 13-year old patient has been investigated since infancy because of a cholestasis phenotype caused by bile-acid insufficiency. Several years ago it was shown that she had a 662C>T missense mutation in AKR1D1 causing a Pro198Leu substitution. It was found that the patient had an almost total absence of 5beta-reduced metabolites of corticosteroids and severely reduced production of 5beta-reduced metabolites of other steroids. The patient is healthy in spite of her earlier hepatic failure and is on no treatment. All her vital signs were normal, as were results of many biochemical analyses. She had normal pubertal changes and experiences regular menstrual cycles. There was no evidence for any clinical condition that could be attributed to attenuated ability to metabolize steroids in normal fashion. Both parents were heterozygous for the mutation but the steroid excretion was entirely normal, although an older female sibling showed definitive evidence for attenuated 5beta-reduction of cortisol. A younger brother had a normal steroid metabolome. The sibling genotypes were not available.

17-Hydroxylase/C17,20-lyase (CYP17) is not the enzyme responsible for side-chain cleavage of cortisol and its metabolites.

The question addressed in this study was the nature of the enzyme required to remove the side-chain of 17-hydroxycorticosteroids, leading in the case of cortisol to the excretion of 11beta-hydroxyandrosterone, 11-oxo-androsterone and the corresponding etiocholanolones. We questioned whether it could be CYP17, the 17-hydroxylase/17,20-lyase utilized in androgen synthesis. The conversion of exogenous cortisol to C(19) steroids in patients with complete 17-hydroxylase deficiency (17HD) was studied rationalizing that if CYP17 was involved no C(19) steroids would be formed. The urinary excretion of the four 11-oxy-C(19) steroids as well as many of the major C(21) cortisol metabolites were measured by GC/MS. Our results showed that the conversion of cortisol to C(19) steroids was normal in 17HD indicating that a currently unidentified enzyme must be responsible for this transformation. A secondary goal was to determine to what extent 11-oxy-C(19) steroids were metabolites of cortisol or adrenal synthesized 11beta-hydroxyandrostenedione. Since cortisol-treated 17HD patients cannot produce androstenedione, all C(19) 11-oxy-metabolites excreted must be derived from exogenous cortisol. The extent to which 17HD patients have lower relative excretion of C(19) steroids should reflect the absence of 11beta-hydroxyandrostenedione metabolites. Our results showed almost all of 11-oxo-etiocholanolone and 11beta-hydroxyetiocholanolone were cortisol metabolites, but in contrast the excretion of 11beta-hydroxyandrosterone was less than 10% that of normal individuals, indicating that in excess of 90% must be a metabolite of 11beta-hydroxyandrostenedione.

Reduced 11beta-hydroxysteroid dehydrogenase type 1 activity in obese boys.

OBJECTIVE: The incidence of childhood obesity and type 2 diabetes has reached epidemic proportions. Glucocorticoid excess causes central obesity and diabetes mellitus as seen in Cushing's syndrome. The 11beta-hydroxysteroid dehydrogenase type 1 enzyme (11beta-HSD1) regenerates active cortisol from inactive cortisone. Altered 11beta-HSD1 may cause tissue-specific Cushing's syndrome with central obesity and impaired glucose homeostasis. DESIGN, PATIENTS, AND METHODS: Clinical and laboratory characteristics, and anthropometric measurements were determined in 15 male and 6 female obese pubertal children (aged 12-18 years, Tanner stages 2-5). In addition, analyses of 24-h excretion rates of glucocorticoids were also performed in 21 age-, sex-, and pubertal stage-matched non-obese children using gas chromatographic-mass spectrometric (GC-MS) analysis. RESULTS: 11beta-HSD1 activity (urinary tetrahydrocortisol (THF) + 5alpha-THF/tetrahydrocortisone (THE) ratio) was lower in obese when compared with non-obese boys. In addition, obese children had a higher total cortisol metabolite excretion than non-obese children. 11beta-HSD1 activity was significantly related to age in lean and obese children. Standard deviation score (SDS)-body mass index did not correlate with 11beta-HSD1 activity, or with total cortisol metabolite excretion within each group. In obese children, 11beta-HSD1 activity and total cortisol metabolite excretion showed no correlation to waist-to-hip ratio, fat mass (percentage of body mass), or the homeostasis model assessment of insulin resistance index. CONCLUSIONS: In conclusion, our findings strongly suggest that 11beta-HSD1 activity increases with age, and is reduced in obese boys. In addition, obese children have a higher total cortisol metabolites excretion suggesting a stimulated hypothalamus-pituitary-adrenal axis.

Modified-Release and Conventional Glucocorticoids and Diurnal Androgen Excretion in Congenital Adrenal Hyperplasia.

Context: The classic androgen synthesis pathway proceeds via dehydroepiandrosterone, androstenedione, and testosterone to 5α-dihydrotestosterone. However, 5α-dihydrotestosterone synthesis can also be achieved by an alternative pathway originating from 17α-hydroxyprogesterone (17OHP), which accumulates in congenital adrenal hyperplasia (CAH). Similarly, recent work has highlighted androstenedione-derived 11-oxygenated 19-carbon steroids as active androgens, and in CAH, androstenedione is generated directly from 17OHP. The exact contribution of alternative pathway activity to androgen excess in CAH and its response to glucocorticoid (GC) therapy is unknown. Objective: We sought to quantify classic and alternative pathway-mediated androgen synthesis in CAH, their diurnal variation, and their response to conventional GC therapy and modified-release hydrocortisone. Methods: We used urinary steroid metabolome profiling by gas chromatography-mass spectrometry for 24-hour steroid excretion analysis, studying the impact of conventional GCs (hydrocortisone, prednisolone, and dexamethasone) in 55 adults with CAH and 60 controls. We studied diurnal variation in steroid excretion by comparing 8-hourly collections (23:00-7:00, 7:00-15:00, and 15:00-23:00) in 16 patients with CAH taking conventional GCs and during 6 months of treatment with modified-release hydrocortisone, Chronocort. Results: Patients with CAH taking conventional GCs showed low excretion of classic pathway androgen metabolites but excess excretion of the alternative pathway signature metabolites 3α,5α-17-hydroxypregnanolone and 11ß-hydroxyandrosterone. Chronocort reduced 17OHP and alternative pathway metabolite excretion to near-normal levels more consistently than other GC preparations. Conclusions: Alternative pathway-mediated androgen synthesis significantly contributes to androgen excess in CAH. Chronocort therapy appears superior to conventional GC therapy in controlling androgen synthesis via alternative pathways through attenuation of their major substrate, 17OHP.

Steroid metabolome analysis reveals prevalent glucocorticoid excess in primary aldosteronism.

BACKGROUND: Adrenal aldosterone excess is the most common cause of secondary hypertension and is associated with increased cardiovascular morbidity. However, adverse metabolic risk in primary aldosteronism extends beyond hypertension, with increased rates of insulin resistance, type 2 diabetes, and osteoporosis, which cannot be easily explained by aldosterone excess. METHODS: We performed mass spectrometry-based analysis of a 24-hour urine steroid metabolome in 174 newly diagnosed patients with primary aldosteronism (103 unilateral adenomas, 71 bilateral adrenal hyperplasias) in comparison to 162 healthy controls, 56 patients with endocrine inactive adrenal adenoma, 104 patients with mild subclinical, and 47 with clinically overt adrenal cortisol excess. We also analyzed the expression of cortisol-producing CYP11B1 and aldosterone-producing CYP11B2 enzymes in adenoma tissue from 57 patients with aldosterone-producing adenoma, employing immunohistochemistry with digital image analysis. RESULTS: Primary aldosteronism patients had significantly increased cortisol and total glucocorticoid metabolite excretion (all P < 0.001), only exceeded by glucocorticoid output in patients with clinically overt adrenal Cushing syndrome. Several surrogate parameters of metabolic risk correlated significantly with glucocorticoid but not mineralocorticoid output. Intratumoral CYP11B1 expression was significantly associated with the corresponding in vivo glucocorticoid excretion. Unilateral adrenalectomy resolved both mineralocorticoid and glucocorticoid excess. Postoperative evidence of adrenal insufficiency was found in 13 (29%) of 45 consecutively tested patients. CONCLUSION: Our data indicate that glucocorticoid cosecretion is frequently found in primary aldosteronism and contributes to associated metabolic risk. Mineralocorticoid receptor antagonist therapy alone may not be sufficient to counteract adverse metabolic risk in medically treated patients with primary aldosteronism. FUNDING: Medical Research Council UK, Wellcome Trust, European Commission.

Impaired 17,20-Lyase Activity in Male Mice Lacking Cytochrome b5 in Leydig Cells.

Androgen and estrogen biosynthesis in mammals requires the 17,20-lyase activity of cytochrome P450 17A1 (steroid 17-hydroxylase/17,20-lyase). Maximal 17,20-lyase activity in vitro requires the presence of cytochrome b5 (b5), and rare cases of b5 deficiency in human beings causes isolated 17,20-lyase deficiency. To study the consequences of conditional b5 removal from testicular Leydig cells in an animal model, we generated Cyb5(flox/flox):Sf1-Cre (LeyKO) mice. The LeyKO male mice had normal body weights, testis and sex organ weights, and fertility compared with littermates. Basal serum and urine steroid profiles of LeyKO males were not significantly different than littermates. In contrast, marked 17-hydroxyprogesterone accumulation (100-fold basal) and reduced testosterone synthesis (27% of littermates) were observed after human chorionic gonadotropin stimulation in LeyKO animals. Testis homogenates from LeyKO mice showed reduced 17,20-lyase activity and a 3-fold increased 17-hydroxylase to 17,20-lyase activity ratio, which were restored to normal upon addition of recombinant b5. We conclude that Leydig cell b5 is required for maximal androgen synthesis and to prevent 17-hydroxyprogesterone accumulation in the mouse testis; however, the b5-independent 17,20-lyase activity of mouse steroid 17-hydroxylase/17,20-lyase is sufficient for normal male genital development and fertility. LeyKO male mice are a good model for the biochemistry but not the physiology of isolated 17,20-lyase deficiency in human beings.

Dual-5α-Reductase Inhibition Promotes Hepatic Lipid Accumulation in Man.

CONTEXT: 5α-Reductase 1 and 2 (SRD5A1, SRD5A2) inactivate cortisol to 5α-dihydrocortisol in addition to their role in the generation of DHT. Dutasteride (dual SRD5A1 and SRD5A2 inhibitor) and finasteride (selective SRD5A2 inhibitor) are commonly prescribed, but their potential metabolic effects have only recently been identified. OBJECTIVE: Our objective was to provide a detailed assessment of the metabolic effects of SRD5A inhibition and in particular the impact on hepatic lipid metabolism. DESIGN: We conducted a randomized study in 12 healthy male volunteers with detailed metabolic phenotyping performed before and after a 3-week treatment with finasteride (5 mg od) or dutasteride (0.5 mg od). Hepatic magnetic resonance spectroscopy (MRS) and two-step hyperinsulinemic euglycemic clamps incorporating stable isotopes with concomitant adipose tissue microdialysis were used to evaluate carbohydrate and lipid flux. Analysis of the serum metabolome was performed using ultra-HPLC-mass spectrometry. SETTING: The study was performed in the Wellcome Trust Clinical Research Facility, Queen Elizabeth Hospital, Birmingham, United Kingdom. MAIN OUTCOME MEASURE: Incorporation of hepatic lipid was measured with MRS. RESULTS: Dutasteride, not finasteride, increased hepatic insulin resistance. Intrahepatic lipid increased on MRS after dutasteride treatment and was associated with increased rates of de novo lipogenesis. Adipose tissue lipid mobilization was decreased by dutasteride. Analysis of the serum metabolome demonstrated that in the fasted state, dutasteride had a significant effect on lipid metabolism. CONCLUSIONS: Dual-SRD5A inhibition with dutasteride is associated with increased intrahepatic lipid accumulation.

Gender-Specific Differences in Skeletal Muscle 11ß-HSD1 Expression Across Healthy Aging.

CONTEXT: Cushing's syndrome is characterized by marked changes in body composition (sarcopenia, obesity, and osteoporosis) that have similarities with those seen in aging. 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) converts glucocorticoids to their active form (cortisone to cortisol in humans), resulting in local tissue amplification of effect. OBJECTIVE: To evaluate 11ß-HSD1 expression and activity with age, specifically in muscle. To determine putative causes for increased activity with age and its consequences upon phenotypic markers of adverse aging. DESIGN: Cross-sectional observational study. SETTING: National Institute for Health Research-Wellcome Trust Clinical Research Facility, Birmingham, United Kingdom. PATIENTS OR OTHER PARTICIPANTS: Healthy human volunteers age 20 to 81 years (n = 134; 77 women, 57 men). INTERVENTIONS: Day attendance at research facility for baseline observations, body composition analysis by dual-energy x-ray absorptiometry, jump-plate mechanography, grip strength analysis, baseline biochemical profiling, urine collection, and vastus lateralis muscle biopsy. MAIN OUTCOME MEASURE(S): Skeletal muscle gene expression, urine steroid profile, bivariate correlations between expression/activity and phenotypic/biochemical variables. RESULTS: Skeletal muscle 11ß-HSD1 expression was increased 2.72-fold in women over 60 years of age compared to those aged 20-40 years; no differences were observed in men. There was a significant positive correlation between skeletal muscle 11ß-HSD1 expression and age in women across the group (rho = 0.40; P = .009). No differences in expression of 11ß-HSD type 2, glucocorticoid receptor, or hexose-6-phosphate dehydrogenase between age groups were observed in either sex. Urinary steroid markers of 11ß-HSD1, 11ß-HSD type 2, or 5α-reductase were similar between age groups. Skeletal muscle 11ß-HSD1 expression was associated with reduced grip strength in both sexes and correlated positively with percentage of body fat, homeostasis model of assessment for insulin resistance, total cholesterol, LH, and FSH and negatively with bone mineral content and IGF-1 in women. CONCLUSIONS: Skeletal muscle 11ß-HSD1 is up-regulated with age in women and is associated with reduced grip strength, insulin resistance, and an adverse body composition profile. Selective inhibition of 11ß-HSD1 may offer a novel strategy to prevent and/or reverse age-related sarcopenia.

5α-Reductase Type 2 Regulates Glucocorticoid Action and Metabolic Phenotype in Human Hepatocytes.

Glucocorticoids and androgens have both been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); androgen deficiency in males, androgen excess in females, and glucocorticoid excess in both sexes are associated with NAFLD. Glucocorticoid and androgen action are regulated at a prereceptor level by the enzyme 5α-reductase type 2 (SRD5A2), which inactivates glucocorticoids to their dihydrometabolites and converts T to DHT. We have therefore explored the role of androgens and glucocorticoids and their metabolism by SRD5A2 upon lipid homeostasis in human hepatocytes. In both primary human hepatocytes and human hepatoma cell lines, glucocorticoids decreased de novo lipogenesis in a dose-dependent manner. Whereas androgen treatment (T and DHT) increased lipogenesis in cell lines and in primary cultures of human hepatocytes from female donors, it was without effect in primary hepatocyte cultures from men. SRD5A2 overexpression reduced the effects of cortisol to suppress lipogenesis and this effect was lost following transfection with an inactive mutant construct. Conversely, pharmacological inhibition using the 5α-reductase inhibitors finasteride and dutasteride augmented cortisol action. We have demonstrated that manipulation of SRD5A2 activity can regulate lipogenesis in human hepatocytes in vitro. This may have significant clinical implications for those patients prescribed 5α-reductase inhibitors, in particular augmenting the actions of glucocorticoids to modulate hepatic lipid flux.

The modulation of corticosteroid metabolism by hydrocortisone therapy in patients with hypopituitarism increases tissue glucocorticoid exposure.

CONTEXT: Patients with hypopituitarism have increased morbidity and mortality. There is ongoing debate about the optimum glucocorticoid (GC) replacement therapy. OBJECTIVE: To assess the effect of GC replacement in hypopituitarism on corticosteroid metabolism and its impact on body composition. DESIGN AND PATIENTS: We assessed the urinary corticosteroid metabolite profile (using gas chromatography/mass spectrometry) and body composition (clinical parameters and full body DXA) of 53 patients (19 female, median age 46 years) with hypopituitarism (33 ACTH-deficient/20 ACTH-replete) (study A). The corticosteroid metabolite profile of ten patients with ACTH deficiency was then assessed prospectively in a cross over study using three hydrocortisone (HC) dosing regimens (20/10 mg, 10/10 mg and 10/5 mg) (study B) each for 6 weeks. 11 beta-hydroxysteroid dehydrogenase 1 (11ß-HSD1) activity was assessed by urinary THF+5α-THF/THE. SETTING: Endocrine Centres within University Teaching Hospitals in the UK and Ireland. MAIN OUTCOME MEASURES: Urinary corticosteroid metabolite profile and body composition assessment. RESULTS: In study A, when patients were divided into three groups - patients not receiving HC and patients receiving HC≤20 mg/day or HC>20 mg/day - patients in the group receiving the highest daily dose of HC had significantly higher waist-to-hip ratio (WHR) than the ACTH replete group. They also had significantly elevated THF+5α-THF/THE (P=0.0002) and total cortisol metabolites (P=0.015). In study B, patients on the highest HC dose had significantly elevated total cortisol metabolites and all patients on HC had elevated THF+5α-THF/THE ratios when compared to controls. CONCLUSIONS: In ACTH-deficient patients daily HC doses of >20 mg/day have increased WHR, THF+5α-THF/THE ratios and total cortisol metabolites. GC metabolism and induction of 11ß-HSD1 may play a pivitol role in the development of the metabolically adverse hypopituitary phenotype.