Women with polycystic ovary syndrome (PCOS) have high circulating androgens, thought to originate from ovaries and adrenals, and frequently suffer from the metabolic syndrome including obesity. However, serum androgens are positively associated with body mass index (BMI) not only in PCOS, but also in simple obesity, suggesting androgen synthesis within adipose tissue. Thus we investigated androgen generation in human adipose tissue, including expression of 17β-hydroxysteroid dehydrogenase (17β-HSD) isozymes, important regulators of sex steroid metabolism. Paired omental and subcutaneous fat biopsies were obtained from 27 healthy women undergoing elective abdominal surgery (age range 30–50 years; BMI 19.7–39.2 kg/m2). Enzymatic activity assays in preadipocyte proliferation cultures revealed effcient conversion of androstenedione to testosterone in both subcutaneous and omental fat. RT-PCR of whole fat and preadipocytes of subcutaneous and omental origin showed expression of 17β-HSD types 4 and 5, but no relevant expression of 17β-HSD types 1, 2, or 3. Microarray analysis confirmed this expression pattern (17β-HSD5>17β-HSD4) and suggested a higher expression of 17β-HSD5 in subcutaneous fat. Accordingly, quantitative real-time RT-PCR showed significantly higher expression of 17β-HSD5 in subcutaneous compared with omental fat (P<0.05). 17β-HSD5 expression in subcutaneous, but not omental, whole fat correlated significantly with BMI (r=0.51, P<0.05). In keeping with these findings, 17β-HSD5 expression in subcutaneous fat biopsies from six women taking part in a weight loss study decreased significantly with weight loss (P<0.05). A role for 17β-HSD5 in adipocyte differentiation was further supported by the observed increase in 17β-HSD5 expression upon differentiation of stromal preadipocytes to mature adipocytes (n=5; P<0.005), which again was higher in cells of subcutaneous origin. Functional activity of 17β-HSD5 also significantly increased with differentiation, revealing a net gain in androgen activation (androstenedione to testosterone) in subcutaneous cultures, contrasting with a net gain in androgen inactivation (testosterone to androstenedione) in omental cultures. Thus, human adipose tissue is capable of active androgen synthesis catalysed by 17β-HSD5, and increased expression in obesity may contribute to circulating androgen excess.
Marcus Quinkler, Binayak Sinha, Jeremy W Tomlinson, Iwona J Bujalska, Paul M Stewart, and Wiebke Arlt
Stuart A Morgan, Laura L Gathercole, Zaki K Hassan-Smith, Jeremy Tomlinson, Paul M Stewart, and Gareth G Lavery
The aged phenotype shares several metabolic similarities with that of circulatory glucocorticoid excess (Cushing’s syndrome), including type 2 diabetes, obesity, hypertension, and myopathy. We hypothesise that local tissue generation of glucocorticoids by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts 11-dehydrocorticosterone to active corticosterone in rodents (corticosterone to cortisol in man), plays a role in driving age-related chronic disease. In this study, we have examined the impact of ageing on glucocorticoid metabolism, insulin tolerance, adiposity, muscle strength, and blood pressure in both wildtype (WT) and transgenic male mice with a global deletion of 11β-HSD1 (11β-HSD1−/−) following 4 months high-fat feeding. We found that high fat-fed 11β-HSD1−/− mice were protected from age-related glucose intolerance and hyperinsulinemia when compared to age/diet-matched WTs. By contrast, aged 11β-HSD1−/− mice were not protected from the onset of sarcopenia observed in the aged WTs. Young 11β-HSD1−/− mice were partially protected from diet-induced obesity; however, this partial protection was lost with age. Despite greater overall obesity, the aged 11β-HSD1−/− animals stored fat in more metabolically safer adipose depots as compared to the aged WTs. Serum analysis revealed both WT and 11β-HSD1−/− mice had an age-related increase in morning corticosterone. Surprisingly, 11β-HSD1 oxo-reductase activity in the liver and skeletal muscle was unchanged with age in WT mice and decreased in gonadal adipose tissue. These data suggest that deletion of 11β-HSD1 in high fat-fed, but not chow-fed, male mice protects from age-related insulin resistance and supports a metabolically favourable fat distribution.
Iwona J Bujalska, Omar M Durrani, Joseph Abbott, Claire U Onyimba, Pamela Khosla, Areeb H Moosavi, Tristan T Q Reuser, Paul M Stewart, Jeremy W Tomlinson, Elizabeth A Walker, and Saaeha Rauz
Glucocorticoids (GCs) have a profound effect on adipose biology increasing tissue mass causing central obesity. The pre-receptor regulation of GCs by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) that activates cortisol from cortisone has been postulated as a fundamental mechanism underlying the metabolic syndrome mediating adipocyte hyperplasia and hypertrophy in the omental (OM) depot. Orbital adipose tissue (OF) is the site of intense inflammation and tissue remodelling in several orbital inflammatory disease states. In this study, we describe features of the GC metabolic pathways in normal human OF depot and compare it with subcutaneous (SC) and OM depots. Using an automated histological characterisation technique, OF adipocytes were found to be significantly smaller (parameters: area, maximum diameter and perimeter) than OM and SC adipocytes (P<0.001). Although immunohistochemical analyses demonstrated resident CD68+ cells in all three whole tissue adipose depots, OF CD68 mRNA and protein expression exceeded that of OM and SC (mRNA, P<0.05; protein, P<0.001). In addition, there was higher expression of glucocorticoid receptor (GR)α mRNA in the OF whole tissue depot (P<0.05). Conversely, 11β-HSD1 mRNA together with the markers of late adipocyte differentiation (FABP4 and G3PDH) were significantly lower in OF. Primary cultures of OF preadipocytes demonstrated predominant 11β-HSD1 oxo-reductase activity with minimal dehydrogenase activity. Orbital adipocytes are smaller, less differentiated, and express low levels of 11β-HSD1 but abundant GRα compared with SC and OM. OF harbours a large CD68+ population. These characteristics define an orbital microenvironment that has the potential to respond to sight-threatening orbital inflammatory disease.
Nikolaos Nikolaou, Anastasia Arvaniti, Nathan Appanna, Anna Sharp, Beverly A Hughes, Dena Digweed, Martin J Whitaker, Richard Ross, Wiebke Arlt, Trevor M Penning, Karen Morris, Sherly George, Brian G Keevil, Leanne Hodson, Laura L Gathercole, and Jeremy W Tomlinson
Steroid 5β-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.
Laura L Gathercole, Nikolaos Nikolaou, Shelley E Harris, Anastasia Arvaniti, Toryn M Poolman, Jonathan M Hazlehurst, Denise V Kratschmar, Marijana Todorčević, Ahmad Moolla, Niall Dempster, Ryan C Pink, Michael F Saikali, Liz Bentley, Trevor M Penning, Claes Ohlsson, Carolyn L Cummins, Matti Poutanen, Alex Odermatt, Roger D Cox, and Jeremy W Tomlinson
Steroid 5β-reductase (AKR1D1) plays important role in hepatic bile acid synthesis and glucocorticoid clearance. Bile acids and glucocorticoids are potent metabolic regulators, but whether AKR1D1 controls metabolic phenotype in vivo is unknown. Akr1d1–/– mice were generated on a C57BL/6 background. Liquid chromatography/mass spectrometry, metabolomic and transcriptomic approaches were used to determine effects on glucocorticoid and bile acid homeostasis. Metabolic phenotypes including body weight and composition, lipid homeostasis, glucose tolerance and insulin tolerance were evaluated. Molecular changes were assessed by RNA-Seq and Western blotting. Male Akr1d1–/– mice were challenged with a high fat diet (60% kcal from fat) for 20 weeks. Akr1d1–/– mice had a sex-specific metabolic phenotype. At 30 weeks of age, male, but not female, Akr1d1–/– mice were more insulin tolerant and had reduced lipid accumulation in the liver and adipose tissue yet had hypertriglyceridemia and increased intramuscular triacylglycerol. This phenotype was associated with sexually dimorphic changes in bile acid metabolism and composition but without overt effects on circulating glucocorticoid levels or glucocorticoid-regulated gene expression in the liver. Male Akr1d1–/– mice were not protected against diet-induced obesity and insulin resistance. In conclusion, this study shows that AKR1D1 controls bile acid homeostasis in vivo and that altering its activity can affect insulin tolerance and lipid homeostasis in a sex-dependent manner.