Exogenous glucocorticoid (GC) therapy, whether physiological replacement in adrenal insufficiency or pharmacological treatment of inflammatory diseases, confers significant cardiometabolic risks, including obesity and insulin resistance. Finding safer alternative GCs is thus a pressing goal. Corticosterone, a minor endogenous GC in humans, may have fewer deleterious metabolic effects than cortisol (hydrocortisone), potentially mediated through selective export via the Abcc1 transporter. In adrenalectomised mice we compared oral corticosterone and cortisol administration and investigated whether Abcc1 mediates differential metabolic effects, and whether such responses are sexually dimorphic. After an initial dose-response study male and female Abcc1-knockout (Abcc1-/-) and wild-type (WT) mice were administered corticosterone or cortisol (25 µg/mL) in drinking water for up to 5 weeks. Cortisol induced greater insulin resistance and elevated energy expenditure compared to corticosterone, an effect more pronounced in male mice. Corticosterone-treated Abcc1-/- males, surprisingly, exhibited reduced fat mass and improved insulin sensitivity compared to WT controls. This protective effect of Abcc1 deficiency was neither observed in cortisol-treated mice nor in female mice receiving either treatment. These findings confirm that even at relatively low doses cortisol drives more severe metabolic dysregulation than corticosterone, independent of Abcc1. Paradoxically Abcc1 deletion protects against corticosterone-induced adiposity in males. Moreover sex modulates GC responses. These findings challenge the presumed role of Abcc1 in GC export and highlight the importance of sex in GC action. Together these results underscore corticosterone's potential as a safer GC therapy, advance our understanding of sex-specific GC action and raise novel questions about Abcc1's role in GC regulation. KEY POINTS: Current glucocorticoid therapies carry significant cardiometabolic risks, including obesity and insulin resistance, highlighting the need for safer alternatives. Corticosterone may have fewer harmful metabolic effects than cortisol, hypothesised to result from its selective export from cells by the Abcc1 transporter. In a mouse model of exogenous glucocorticoid excess cortisol induced more severe insulin resistance and increased energy expenditure compared to corticosterone, but only in male mice. Unexpectedly Abcc1-knockout males treated with corticosterone showed reduced fat mass and improved insulin sensitivity, an effect not seen with cortisol or in females. These results challenge the assumed role of Abcc1 in glucocorticoid export, highlight sex differences in glucocorticoid action and support corticosterone's potential as a safer therapeutic option.

Pancreatic cancer (PC) represents one of the biggest challenges in terms of cancer treatment, mainly due to its continuously rising incidence, advanced stage at time of diagnosis, and dismal 5-year overall survival, which has not improved in recent decades despite the major advances made in oncological therapies. The limited progress in developing more effective therapies is, in part, attributable to the vast desmoplastic stroma present in PC. Additionally, immunosuppressive steroid-signalling has recently been shown to aid the development and metastasis of various tumour types. Therefore, we sought to explore whether local steroidogenesis and steroid signalling within the tumour microenvironment (TME) play a role in pancreatic cancer development. Reanalysis of publicly available datasets, including single cell RNA sequencing, as well as in vivo metastatic pancreatic ductal adenocarcinoma (PDAC) mouse models, allowed us to identify Hsd11b1 as the key enzyme responsible for locally elevated levels of the immunosuppressive glucocorticoid hormone, corticosterone. We identified fibroblasts as the major Hsd11b1-expressing populations in the pancreatic TME. Specifically, in mice, Hsd11b1 expression is primarily observed in iCAFs. Additionally, we found that patients with higher HSD11B1 expression present an increased mortality rate as well as an enriched fibrotic signature and inhibited immune activity. Collectively, these findings suggest that Hsd11b1 upregulation in iCAFs could be aiding PDAC development by promoting the activation of glucocorticoids directly in the TME. The presence of glucocorticoids inhibits inflammation and could also be enhancing local fibrosis by autocrine signalling in the fibroblast population. Given the urgent need for effective treatments in this fatal disease, targeting HSD11B1 represents a promising therapeutic strategy to overcome the immunosuppressive desmoplastic barrier and improve patient outcomes in pancreatic cancer. ### Competing Interest Statement The authors have declared no competing interest. CRUK, RCCFEL\100095 NSF-BIO/UKRI-BBSRC, BB/V006126/1 MRC, MR/V028995/1

BACKGROUND AND PURPOSE:The contribution of obesity to pulmonary arterial hypertension (PAH) pathophysiology remains poorly understood. Adipose tissue synthesises estrogens via cytochrome P450 (CYP) 19A1 (aromatase), whereas circulating estrogens are metabolised in the lung by CYP1A1. This study investigated whether obesity predisposes to PAH through enhanced estrogen synthesis and metabolism. EXPERIMENTAL APPROACH:A normoxic, two-hit, rat model of obesity-associated pulmonary hypertension (PH) was developed, combining Sugen 5416 (Sugen, Su) with a high-fat diet (HFD). Estrogen levels in SuHFD rat plasma and epicardial adipose tissue (EAT) from PAH patients were quantified using LC-MS/MS. CYP1A1 expression was assessed in lung and cardiac adipose tissue from SuHFD rats and PAH patients. The therapeutic potential of the CYP1A1 inhibitor hesperetin was evaluated in vivo. Complementary studies used pulmonary artery smooth muscle cells (PASMCs) from PAH patients and Simpson-Golabi-Behmel syndrome (SGBS) adipocytes. KEY RESULTS:HFD-fed rats of both sexes developed mild PH, which Sugen moderately exacerbated. EAT from PAH patients exhibited up-regulated aromatase and CYP1A1 expression, along with elevated estrogen levels. Circulating estrone was increased in male SuHFD rats. Pulmonary CYP1A1 expression was elevated in SuHFD rats and PAH patients. Hesperetin attenuated obesity-associated PH, reducing CYP1A1 expression in SuHFD rat lungs and PAH PASMCs. CYP1A1 induction in female SuHFD rat pericardial adipose tissue and Sugen-treated SGBS adipocytes was also tempered. CONCLUSION AND IMPLICATIONS:These findings implicate augmented estrogen production by adipose tissue and elevated pulmonary CYP1A1 expression in the pathogenesis of obesity-associated PH. CYP1A1 may represent a novel therapeutic target in obese PAH patients.

We previously published the DexFEM trial, which showed that in women with heavy menstrual bleeding (HMB), oral dexamethasone reduces menstrual blood loss. Here, we report a pharmacodynamic analysis exploring the likely mechanism for this effect. We studied oral dosing with dexamethasone during the mid-luteal phase of two menstrual cycles (1.5 mg daily, 5 days) in five women with HMB (six recruited aged 41–50 years, one withdrew before treatment). Steroid hormones were profiled in serum and endometrium by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We found that following oral dosing, dexamethasone reached the endometrium and that, compared to the preceding control cycle, cortisol (active), cortisone (inactive) and intermediate 11-deoxycortisol were reduced in all samples assessed, both endometrial (n = 4) and serum (n = 5). Concentrations of androgens, androstenedione and testosterone were reduced in serum but not in all tissue samples. This proof-of-concept pharmacodynamic study supports the inference that dexamethasone is effective in HMB by altering endometrial glucocorticoid concentrations.

Mammary adipocyte size reflects both local excess of adiposity and adipose tissue dysfunction relevant to breast cancer biology. To identify modifiable factors that are associated with both traditional adiposity indicators and mammary adipocyte size in women with breast cancer, and to compare the individual and simultaneous effect of these factors. Data were collected prospectively from 160 consecutive breast cancer patients (biobank of a breast cancer reference center): factors that may influence body weight and composition (telephone interview), dietary intakes (DHQ-I) and adiposity measurements (anthropometric indices and mammary adipocyte size). Relationships between determinants of adiposity identified in the literature were summarized in a directed acyclic graph. Principal component analysis was conducted to capture dietary intakes from major nutrient intakes. Robust univariate and multivariable linear regression models were used to estimate the associations. Menopausal status, ever smoking, tumour grade and higher weight at 18 years old were consistently associated with higher adiposity. Higher animal fat intakes was consistently associated with higher body mass index (BMI). High educational attainment was consistently associated with lower BMI and waist-to-height ratio. Higher physical activity was associated with lower adiposity and adipocyte cell size, whereas higher age was associated with higher adiposity and adipocyte cell size only in univariate models. Only menopausal status was consistently associated with higher mammary adipocyte size. While excess adiposity is a complex condition that cannot be attributed to a single factor, menopausal status seems to be the main determinant of excess adiposity in women with breast cancer and the only independent determinant of mammary adipocyte size. Among lifestyle factors, ever smoking was the strongest independent determinant of higher adiposity, followed by high intakes of fats, particularly animal fats. If targeted efficiently, some of these modifiable factors could reduce the burden among breast cancer patients.

Background: Data are scarce on steroid hormone variations occurring in adipose tissue (AT) compartments of men and women following weight loss (WL).Objective: Characterise AT function markers, AT steroid hormones, and their change after surgery-induced WL.Methods: AT function markers, fourteen steroid hormones, and steroid-converting enzymes were investigated in omental and subcutaneous AT in 38 participants before and after sleeve gastrectomy.Results: Following WL, adipocyte size as well as AKR1C2, AKR1C3, HSD11B1, SRD5A1 and SRD5A3 expression decreased in both compartments. AT testosterone and 5α-dihydrotestosterone increased in men, and oestrogens decreased in both sexes. Aromatase expression was higher in men before WL and decreased in both sexes. DHRS11 expression decreased in women. 11KDHT was the most concentrated active androgen in women but was not related to depot or WL.Conclusions: Substantial changes occur in AT function markers, steroid hormones, and enzyme expression in abdominal fat compartments in response to surgery-induced WL.

Context Body fat distribution is a risk factor for obesity-associated comorbidities, and adipose tissue dysfunction plays a role in this association. In humans, there is a sex difference in body fat distribution, and steroid hormones are known to regulate several cellular processes within adipose tissue. Objective Our aim was to investigate if intra-adipose steroid concentration and expression or activity of steroidogenic enzymes were associated with features of adipose tissue dysfunction in individuals with severe obesity. Methods Samples from 40 bariatric candidates (31 women, 9 men) were included in the study. Visceral (VAT) and subcutaneous adipose tissue (SAT) were collected during surgery. Adipose tissue morphology was measured by a combination of histological staining and semi-automated quantification. Following extraction, intra-adipose and plasma steroid concentrations were determined by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Aromatase activity was estimated using product over substrate ratio, while AKR1C2 activity was measured directly by fluorogenic probe. Gene expression was measured by quantitative PCR. Results VAT aromatase activity was positively associated with VAT adipocyte hypertrophy (P value(adj) < 0.01) and negatively with plasma high-density lipoprotein (HDL)-cholesterol (P value(adj) < 0.01), while SAT aromatase activity predicted dyslipidemia in women even after adjustment for waist circumference, age, and hormonal contraceptive use. We additionally compared women with high and low visceral adiposity index (VAI) and found that VAT excess is characterized by adipose tissue dysfunction, increased androgen catabolism mirrored by increased AKR1C2 activity, and higher aromatase expression and activity indices. Conclusion In women, increased androgen catabolism or aromatization is associated with visceral adiposity and adipose tissue dysfunction.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Background: Dihydrotestosterone (DHT), the most potent natural androgen, inhibits adipocyte differentiation and likely plays a role in body fat distribution. Human adipose tissue (AT) possesses the enzymatic machinery to convert 11OH-adrenal precursors to 11-ketoDHT (11KDHT), a steroid shown to have androgenic activity similar to that of DHT. Our aim was to determine if AT contains biologically relevant 11-oxygenated androgens levels. Methods: Institutional approval and consent were obtained. Subcutaneous (SAT) and visceral (VAT) adipose tissues were collected in men (n=15) and women (n=23) undergoing sleeve gastrectomy. A validated liquid chromatography-tandem mass spectrometry method was developed to determine steroid hormone amounts in AT. Results: Participants had a preoperative mean BMI of 53 (range 38-65 kg/m2). We detected 11-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), 11-hydroxytestosterone (11OHT), 11-ketotestosterone (11KT) and 11KDHT in AT. The most abundant was 11OHA4, with concentrations varying from 1404 to 20260 pmol/kg, followed by 11KA4, with concentrations varying from 128 to 3711 pmol/kg. VAT had higher amounts of these precursor steroids compared to SAT (p<0.05). 11OHT (range: 85-580 pmol/kg) and 11KT (range: 109-3827 pmol/kg) were present in lower amounts. 11KDHT was quantified in both SAT and VAT and we detected no difference between sexes. The concentration of 11KDHT varied between 111 and 2991 pmol/kg, which is nearly 2-fold higher than DHT (p<0.0001). BMI and adipocyte cell size were not associated with steroid amounts. The waist-to-hip ratio was positively associated with concentrations of testosterone, DHT, 11OHA4, 11OHT, androstenedione and 11KDHT in VAT, and with testosterone and 11OHT in SAT (p<0.05). Conclusions: For the first time, 11-oxygenated androgens were detected in SAT and VAT of both men and women. AT contains more 11KDHT than DHT. Future studies are needed to determine the role of 11-oxygenated androgens in AT physiology and body fat distribution.

46 Histomorphometric analyses of adipose tissue usually require formalin fixation of fresh 47 samples. Our objective was to determine if intact, flash-frozen whole adipose tissue 48 samples stored at -80°C could be used for measurements developed for fresh-fixed 49 adipose tissues. Portions of adipose tissue samples were either formalin-fixed 50 immediately upon sampling or flash-frozen and stored at -80°C and then formalin-fixed 51 during the thawing process. Mean adipocyte diameter was measured. 52 Immunohistochemistry was performed on additional samples to identify macrophage 53 subtypes (M1, CD14+ and M2, CD206+) and total (CD68+) number. All slides were 54 counterstained using haematoxylin & eosin (H&E). Visual inspection of H&E-stained 55 adipose tissue slides performed in a blinded fashion showed little or no sign of cell 56 breakage in 74% of frozen-fixed samples and in 68% of fresh-fixed samples (p>0.5). 57 There was no difference in the distribution frequencies of adipocyte sizes in fresh-fixed 58 vs. frozen-fixed tissues in both depots (p>0.9). Mean adipocyte size from frozen-fixed 59 samples correlated significantly and positively with adipocyte size from fresh-fixed 60 samples (r=0.74, p<0.0001, for both depots). The quality of staining/immunostaining and 61 appearance of tissue architecture were comparable in fresh-fixed vs. frozen-fixed 62 samples. In conclusion, intact flash-frozen adipose tissue samples stored at -80°C can be 63 used to perform techniques conventionally applied to fresh-fixed samples. This approach 64 will allow retrospective studies with frozen human adipose tissue samples. 65 HACB-D-17-00181R2, page 3

Background Obesity fosters worse clinical outcomes in both premenopausal and postmenopausal women with breast cancer. Emerging evidence suggests that an android body fat distribution in particular is deleterious for breast cancer prognosis. The extent of adipose tissue dysfunction, especially how it relates to breast cancer prognostic factors and anthropometric measurements, has not been fully investigated. Objective Our objective was to examine if markers of adipose tissue dysfunction, such as hypertrophy and macrophage accumulation, are relevant for the pathophysiology of breast cancer and its associated prognostic factors in a well-characterised cohort of women with breast cancer who did not receive treatment before surgery. Methods A consecutive series of 164 women with breast cancer provided breast adipose tissue sample. Multivariate generalised linear models were used to test associations of anthropometric indices and prognostic factors with markers of adipose tissue dysfunction. Results We found associations of breast adipocyte size and macrophage infiltration (number of CD68+ cells/100 adipocytes) with adiposity, particularly a strong association between breast adipocyte size and central obesity, independent of total adiposity, age and menopausal status ( β adj = 0.87; p = 0.0001). We also identified relationships of adipocyte hypertrophy and macrophage infiltration with prognostic factors, such as cancer stage and tumour grade ( p < 0.05). RNA expression of pro-inflammatory cytokines ( IL6 , TNF ) and leptin was also increased as a function of adipocyte size and CD86+/CD11c+ macrophage number/100 adipocytes ( p < 0.05). Conclusions Our findings support the model of dysfunctional adipose tissue in obesity-associated breast cancer.

AbstractOxylipins and endocannabinoids (eCBs) both belong to superclasses of lipid mediators with potent inflammation modulatory activities. The adipose tissue (AT) plays a key role in metabolic syndrome-related inflammation, via altered adipocyte physiology, infiltrated macrophages and altered profile of eCBs. We previously reported that DHA is more potent than EPA at modulating systemic inflammation, but the underlying mechanisms remain unclear. The objective of this study was to compare the individual effect of high-dose DHA and of EPA on circulating lipid mediators, i.e. plasma oxylipins and eCBs, and AT-related inflammation. In a randomized double-blind crossover trial, 154 volunteers with abdominal obesity and low-grade inflammation were subjected to three 10-wk supplementation phases: 1- EPA (2.7 g/d); 2- DHA (2.7 g/d); 3- corn oil (control), each separated by a 9-wk washout. Supplements were provided as re-esterified triacylglycerols. Profiling of plasma oxylipins and eCBs was performed on 58 subjects after each phase. Abdominal subcutaneous AT biopsies were also obtained from 13 individuals after each phase. Plasma DHA-, EPA-, arachidonic acid-derived oxylipins were analyzed by LC-MS. eCBs and some of their bioactive congeners were analyzed in plasma and AT by LC-APCI-MS. Adipocyte diameter was determined by histological analysis and AT macrophage infiltration was quantified by double immunofluorescence. Compared with EPA, DHA increased plasma levels of hydroxy-docosahexaenoic acids (7-, 11-, 14-, 4-, 17-HDOHE; P < 0.0001) and palmitoylethanolamide (PEA; P = 0.04). Compared with DHA, EPA led to higher plasma level of hydroxy-eicosapentaenoic acids (12-, 15-, 5-HEPE; P < 0.0001). In the AT, EPA increased the level of oleoylethanolamide (OEA; P = 0.01) compared with DHA, but no other difference was observed between treatments in adipose eCBs and eCB-related lipids. DHA and EPA did not differentially modify adipocyte size distributions (P > 0.50) and proportions of M1-type and M2-type macrophages (P > 0.30). In conclusion, increased plasma levels of anti-inflammatory DHA-derived oxylipins and plasma PEA may be responsible, at least to some extent, for the more potent anti-inflammatory effects of DHA compared with EPA observed in the ComparED study. Conversely, subcutaneous AT does not seem to be involved in explaining such differences between EPA and DHA.

The presence of estrogens, androgens and glucocorticoids as well as their receptors and steroid converting enzymes in adipose tissue has been established. Their contribution to diseases such as obesity, diabetes and hormone-dependent cancers is an active area of research. Our objective was to develop a LC-MS/MS method to quantify bioactive estrogens and glucocorticoids simultaneously in human adipose tissue. Estrogens and glucocorticoids were extracted from adipose tissue samples using solid-phase extraction. Estrogens were derivatized using 1-(2,4-dinitro-5-fluorophenyl)-4-methylpiperazine (PPZ) and methyl iodide to generate a permanently charged molecule (MPPZ). Steroids were separated and quantified by LC-MS/MS. The limit of quantitation for the steroids was between 15 and 100 pg per sample. Accuracy and precision were acceptable (< 20%). Using this method, estradiol, estrone, cortisone and cortisol were quantified in adipose tissue from women with and without breast cancer. This novel assay of estrogens and glucocorticoids by LC-MS/MS coupled with derivatization allowed simultaneous quantification of a panel of steroids in human adipose tissue across the endogenous range of concentrations encountered in health and disease.

Abstract Context Adipose tissue is an important site for extragonadal steroid hormone biosynthesis through the expression and activity of P450 aromatase, 11β-hydroxysteroid dehydrogenase (HSD) 1, and 17β-HSDs. The contribution of steroid hormones produced by adjacent adipose tissue for the progression and survival of breast tumors is unknown. Objective To quantify estrogens (estradiol, estrone) and glucocorticoids (cortisol, cortisone) in breast adipose tissue from both healthy and diseased women and their relationships with adiposity indices and breast cancer prognostic markers. Design and setting Breast adipose tissue was collected at time of surgery. Patients Pre- and postmenopausal women undergoing partial mastectomy for treatment of breast cancer (n = 17) or reduction mammoplasty (n = 6) were studied. Interventions Relative estrogen and glucocorticoid amounts were determined by liquid chromatography tandem mass spectrometry. Results The targeted steroids were reliably detected and quantified in mammary adipose tissues. Women with ER+/PR+ tumor had higher relative estradiol amount than women with ER–/PR– tumor (P < .05). The ratio of estradiol-to-estrone was higher in lean women than in women with a body mass index (BMI) ≥ 25 kg/m2 (P < .05). Mixed-model analyses showed that estradiol, cortisone, and cortisol were negatively associated with tumor size (P < .05). Relationships between glucocorticoids and tumor size remained significant after adjustment for BMI. The cortisol-to-cortisone ratio was negatively associated with tumor stage (P < .05) independently of BMI. Conclusions We reliably quantified estrogens and glucocorticoids in breast adipose tissue from healthy women and women suffering from breast cancer. Our findings suggest that smaller breast tumors are associated with higher relative amounts of estradiol and cortisol in adipose tissue.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Histomorphometric analyses of adipose tissue usually require formalin fixation of fresh samples. Our objective was to determine if intact, flash-frozen whole adipose tissue samples stored at − 80 °C could be used for measurements developed for fresh-fixed adipose tissues. Portions of adipose tissue samples were either formalin-fixed immediately upon sampling or flash-frozen and stored at − 80 °C and then formalin-fixed during the thawing process. Mean adipocyte diameter was measured. Immunohistochemistry was performed on additional samples to identify macrophage subtypes (M1, CD14 + and M2, CD206 +) and total (CD68 +) number. All slides were counterstained using haematoxylin and eosin (H&E). Visual inspection of H&E-stained adipose tissue slides performed in a blinded fashion showed little or no sign of cell breakage in 74% of frozen-fixed samples and in 68% of fresh-fixed samples (p > 0.5). There was no difference in the distribution frequencies of adipocyte sizes in fresh-fixed vs. frozen-fixed tissues in both depots (p > 0.9). Mean adipocyte size from frozen-fixed samples correlated significantly and positively with adipocyte size from fresh-fixed samples (r = 0.74, p < 0.0001, for both depots). The quality of staining/immunostaining and appearance of tissue architecture were comparable in fresh-fixed vs. frozen-fixed samples. In conclusion, intact flash-frozen adipose tissue samples stored at − 80 °C can be used to perform techniques conventionally applied to fresh-fixed samples. This approach allows for retrospective studies with frozen human adipose tissue samples.