Gender-specific links between hepatic 11beta reduction of cortisone and adipokines

Objective: Reduction of cortisone to cortisol is mediated by 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1), a putative key enzyme in obesity‐related complications. Experimental studies suggest that adipokines, notably leptin and tumor necrosis factor‐α (TNF‐α), are of importance for 11βHSD1 activity. We hypothesized that the regulation of hepatic preceptor glucocorticoid metabolism is gender‐specific and associated with circulating levels of leptin and TNF‐α receptors and/or sex hormones. Research Methods and Procedures: A total of 34 males and 38 women (14 premenopausal and 22 postmenopausal) underwent physical examination and fasting blood sampling. Insulin sensitivity was tested by euglycemic hyperinsulinemic clamps, and hepatic 11βHSD1 enzyme activity was estimated by the conversion of orally‐ingested cortisone to cortisol. Results: Hepatic 11βHSD1 activity was negatively associated with leptin and soluble TNF (sTNF) r1 and sTNFr2 in males. These correlations remained significant after adjustment for age and insulin sensitivity, and for sTNF‐α receptors also after adjustment of BMI and waist circumference. In contrast, 11β reduction of cortisone was positively associated to leptin in females after adjustment for BMI and waist circumference. Discussion: Hepatic 11β reduction shows different links to circulating adipocyte‐derived hormones in males and females. This emphasizes the need for further studies on tissue‐specific regulation of 11βHSD1 in both genders.     

Molecular Screening of the 11β‐HSD1 Gene in Men Characterized by the Metabolic Syndrome

Adipose tissue type 1 11β‐hydroxysteroid dehydrogenase (11β‐HSD1), which generates hormonally active cortisol from inactive cortisone, has been shown to play a central role in adipocyte differentiation and abdominal obesity‐related metabolic complications. The objective was to investigate whether genetic variations in the human 11β‐HSD1 gene are associated with the metabolic syndrome among French‐Canadian men. We sequenced all exons, the exon‐intron splicing boundaries, and 5′ and 3′ regions of the human 11β‐HSD1 gene in 36 men with the metabolic syndrome, as defined by the National Cholesterol Education Program‐Adult Treatment Panel III, and two controls. Three intronic sequence variants were identified: two single‐nucleotide polymorphisms in intron 3 (g.4478T>G) and intron 4 (g.10733G>C) and one insertion in intron 3 (g.4437‐4438insA). The relative allele frequency was 19.6%, 22.1%, and 19.6% for the g.4478G, g.10733C, and g.4438insA alleles, respectively. One single‐nucleotide polymorphism was identified in exon 6 (c.744G>C or G248G). The frequency of the c.744C allele was only 0.46% in a sample of 217 men. Variants were not associated with components of the metabolic syndrome except for plasma apolipoprotein B levels. In conclusion, molecular screening of the 11β‐HSD1 gene did not reveal any sequence variations that can significantly contribute to the etiology of the metabolic syndrome among French‐Canadians.     

Calcium and 1,25-dihydroxyvitamin D3 regulation of adipokine expression

Objective: Obesity is associated with elevated oxidative stress and low‐grade systemic inflammation. We have demonstrated recently that 1α,25‐(OH)₂‐D₃ promotes reactive oxygen species production in cultured adipocytes, whereas suppression of 1α,25‐(OH)₂‐D₃ by increasing dietary calcium down‐regulates diet‐induced oxidative stress in aP2‐agouti transgenic mice. However, whether the anti‐obesity effect of dietary calcium plays a role in regulation of obesity‐associated inflammation is not clear. Research Methods and Procedures: We investigated the role of dietary calcium in the regulation of inflammatory cytokine production in aP2‐agouti transgenic mice fed low‐ and high‐calcium obesigenic diets and in the modulation of cytokine production by 1α,25‐(OH)₂‐D₃ in cultured murine and human adipocytes. Results: The high‐calcium diet inhibited the expression of pro‐inflammatory factors tumor necrosis factor α and interleukin (IL)‐6 by 64% and 51%, respectively (p < 0.001), in visceral fat, stimulated the expression of the anti‐inflammatory factors IL‐15 and adiponectin by 52% (p = 0.001) and 54% (p = 0.025), respectively, in visceral fat, and induced a 2‐fold increase in IL‐15 expression in soleus muscle (p = 0.01) compared with litter mate controls on a low‐calcium diet. 1α,25‐(OH)₂‐D₃ also markedly stimulated the expression of tumor necrosis factor α (p < 0.001) and IL‐6 (p = 0.016) in differentiated 3T3‐L1 adipocytes and increased IL‐6 (p = 0.004) and IL‐8 (p < 0.001) production in differentiated human adipocytes. These effects were blocked by calcium channel antagonism with nifedipine. Discussion: These data demonstrate that 1α,25‐(OH)₂‐D₃ favors inflammatory cytokine expression and inhibits anti‐inflammatory cytokine expression; accordingly, suppression of 1α,25‐(OH)₂‐D₃ by dietary calcium inhibits adipocyte‐derived inflammation associated with obesity.     

1Alpha,25-dihydroxyvitamin D3 modulation of adipocyte reactive oxygen species production

Objective: We have previously shown 1α,25‐dihydroxyvitamin D₃ [1α,25‐(OH)₂D₃] to inhibit mitochondrial uncoupling protein 2 (UCP2) expression in adipocytes and that in vivo suppression of calcitriol levels with calcium‐rich diets increases UCP2 expression. Because UCP2 plays a significant role in the clearance of reactive oxygen species (ROS), we studied the effect of calcitriol on ROS production and ROS‐induced adipocyte proliferation. Research Methods and Procedures: ROS production in human and murine adipocytes was stimulated by high glucose (30 mM) or H₂O₂ (100 nM). Results: Both approaches resulted in increased ROS production by 27% to 100% (p < 0.05) and increased cell proliferation by 15% to 39% (p < 0.03). These effects were augmented by the addition of mitochondrial uncoupling inhibitor guanosine 5′‐diphosphate (GDP; 100 μM) or 1α,25‐(OH)₂D₃ (10 nM) and attenuated by UCP2 overexpression, suggesting that inhibition of mitochondrial uncoupling suppresses clearance of ROS and increases adipocyte proliferation. The addition of α ± tocopherol (1 μM) inhibited cell proliferation in adipocytes treated with either H₂O₂ or high glucose, indicating that ROS plays a major role in the regulation of cell proliferation in adipocytes. Moreover, stimulation of ROS with high glucose and H₂O₂ resulted in a 2‐ to 5‐fold increase in adipocyte intracellular calcium ([Ca²⁺]i; p < 0.001), and calcium channel antagonism (nifedipine, 10 μM) suppressed ROS induced calcium influx and cell proliferation, indicating that [Ca²⁺]i may also regulate ROS production and exert a mitogenic effect in adipocytes. Discussion: These data support a role of 1α,25‐(OH)₂D₃, UCP2, and [Ca²⁺]i in the regulation of adipocyte ROS production.     

Cortisone induces insulin resistance in C2C12 myotubes through activation of 11beta‐hydroxysteroid dehydrogenase 1 and autocrinal regulation

The enzyme 11β‐hydroxysteroid dehydrogenase 1 (11β‐HSD1) is known to catalyse inactive glucocorticoids into active forms, and its dysregulation in adipose and muscle tissues has been implicated in the development of metabolic syndrome. To delineate the molecular mechanism by which active cortisol has an antagonizing effect against insulin, we optimized the metabolic production of cortisol and its biological functions in myotubes (C2C12). Myotubes supplemented with cortisone actively catalysed its conversion into cortisol, which in turn abolished phosphorylation of Akt in response to insulin treatment. This led to diminished uptake of insulin‐induced glucose. This was corroborated by the application of 11β‐HSD1 inhibitor glycyrrhetinic acid and a glucocorticoid receptor antagonist RU‐486, which reversed completely the antagonizing effects of cortisol on insulin action. Therefore, development of specific inhibitors targeting 11β‐HSD1 might be a promising way to improve impaired insulin‐stimulated glucose uptake. Copyright © 2013 John Wiley & Sons, Ltd.     

Sex-specific difference in the interconversion of cortisol and cortisone in men and women

Objective: Our objective was to demonstrate that the smaller oxoreductase activity of 11β‐HSD1 in women would shift the interconversion of cortisol and cortisone toward cortisone, resulting in a larger amount of generated labeled cortisone in healthy women than in healthy men. Research Methods and Procedures: Using mass spectrometry, the amount of cortisone generated from a continuous infusion (8 am to 6 pm ) of stable‐labeled cortisol (1α,2α‐d‐cortisol) was determined in non‐obese and in obese (BMI >35 kg/m²) men and women during steady‐state conditions (from 2 pm to 6 pm ). In this setting, the amount of generated labeled cortisone (expressed as % of the achieved steady‐state concentrations of labeled cortisol) reflects the sum of the bi‐directional conversion of cortisol into cortisone (and vice versa) by 11β‐hydroxysteroid dehydrogenase. Results: The amount of generated labeled cortisone was higher in men than in women (p < 0.0001). This sex difference was higher in obese than in non‐obese patients (p = 0.0062). Conclusions: The interconversion of cortisol and cortisone during steady‐state conditions is shifted toward cortisol in men as compared with women. This suggests a higher overall oxoreductase activity of 11β‐hydroxysteroid dehydrogenase type 1 in men than in women. This sex‐specific difference is maintained in obesity.     

Omental 11beta-hydroxysteroid dehydrogenase 1 correlates with fat cell size independently of obesity

Objectives: In ideopathic obesity, there is evidence that enhanced cortisol regeneration within abdominal subcutaneous adipose tissue may contribute to adiposity and metabolic disease. Whether the cortisol regenerating enzyme, 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1), or glucocorticoid receptor (GRα) levels are altered in other adipose depots remains uncertain. Our objective was to determine the association between 11βHSD1 and GRα mRNA levels in four distinct adipose depots and measures of obesity and the metabolic syndrome. Research Methods and Procedures: Adipose tissue biopsies were collected from subcutaneous (abdominal, thigh, gluteal) and intra‐abdominal (omental) adipose depots from 21 women. 11βHSD1 and GRα mRNA levels were measured by real‐time polymerase chain reaction. Body composition, fat distribution, fat cell size, and blood lipid, glucose, and insulin levels were measured. Results: 11βHSD1 mRNA was highest in abdominal subcutaneous (p < 0.001) and omental (p < 0.001) depots and was positively correlated with BMI and visceral adiposity in all depots. Omental 11βHSD1 correlated with percent body fat (R = 0.462, p < 0.05), fat cell size (R = 0.72, p < 0.001), and plasma triglycerides (R = 0.46, p < 0.05). Conversely, GRα mRNA was highest in omental fat (p < 0.001). GRα mRNA was negatively correlated with BMI in the abdominal subcutaneous (R = ?0.589, p < 0.05) and omental depots (R = ?0.627, p < 0.05). Omental GRα mRNA was inversely associated with visceral adiposity (R = ?0.507, p < 0.05), fat cell size (R = ?0.52, p < 0.01), and triglycerides (R = ?0.50, p < 0.05). Discussion: Obesity was associated with elevated 11βHSD1 mRNA in all adipose compartments. GRα mRNA is reduced in the omental depot with obesity. The novel correlation of 11βHSD1 with omental fat cell size, independent of obesity, suggests that intracellular cortisol regeneration is a strong predictor of hypertrophy in the omentum.     

Mutations of key hydrophobic surface residues of 11β‐hydroxysteroid dehydrogenase type 1 increase solubility and monodispersity in a bacterial expression system

11β‐Hydroxysteroid dehydrogenase type 1 (11β‐HSD1) is a key enzyme in the conversion of cortisone to the functional glucocorticoid hormone cortisol. This activation has been implicated in several human disorders, notably the metabolic syndrome where 11β‐HSD1 has been identified as a novel target for potential therapeutic drugs. Recent crystal structures have revealed the presence of a pronounced hydrophobic surface patch lying on two helices at the C‐terminus. The physiological significance of this region has been attributed to facilitating substrate access by allowing interactions with the endoplasmic reticulum membrane. Here, we report that single mutations that alter the hydrophobicity of this patch (I275E, L266E, F278E, and L279E in the human enzyme and I275E, Y266E, F278E, and L279E in the guinea pig enzyme) result in greatly increased yields of soluble protein on expression in E. coli. Kinetic analyses of both reductase and dehydrogenase reactions indicate that the F278E mutant has unaltered K_m values for steroids and an unaltered or increased k_cat. Analytical ultracentrifugation shows that this mutation also decreases aggregation of both the human and guinea pig enzymes, resulting in greater monodispersity. One of the mutants (guinea pig F278E) has proven easy to crystallize and has been shown to have a virtually identical structure to that previously reported for the wild‐type enzyme. The human F278E enzyme is shown to be a suitable background for analyzing the effects of naturally occurring mutations (R137C, K187N) on enzyme activity and stability. Hence, the F278E mutants should be useful for many future biochemical and biophysical studies of the enzyme.     

Depot-specific regulation of the conversion of cortisone to cortisol in human adipose tissue

Objective: Our main objective was to compare the regulation of cortisol production within omental (Om) and abdominal subcutaneous (Abd sc) human adipose tissue. Methods and Procedures: Om and Abd sc adipose tissue were obtained at surgery from subjects with a wide range of BMI. Hydroxysteroid dehydrogenase (HSD) activity (³H‐cortisone and ³H‐cortisol interconversion) and expression were measured before and after organ culture with insulin and/or dexamethasone. Results: Type 1 HSD (HSD1) mRNA and reductase activity were mainly expressed within adipocytes and tightly correlated with adipocyte size within both depots. There was no depot difference in HSD1 expression or reductase activity, while cortisol inactivation and HSD2 mRNA expression (expressed in stromal cells) were higher in Om suggesting higher cortisol turnover in this depot. Culture with insulin decreased HSD reductase activity in both depots. Culture with dexamethasone plus insulin compared to insulin alone increased HSD reductase activity only in the Om depot. This depot‐specific increase in reductase activity could not be explained by an alteration in HSD1 mRNA or protein, which was paradoxically decreased. However, in Om only, hexose‐6‐phosphate dehydrogenase (H6PDH) mRNA levels were increased by culture with dexamethasone plus insulin compared to insulin alone, suggesting that higher nicotinamide adenine dinucleotide phosphate‐oxidase (NADPH) production within the endoplasmic reticulum (ER) contributed to the higher HSD reductase activity. Discussion: We conclude that in the presence of insulin, glucocorticoids cause a depot‐specific increase in the activation of cortisone within Om adipose tissue, and that this mechanism may contribute to adipocyte hypertrophy and visceral obesity.     

Mammary adipocytes bioactivate 25‐hydroxyvitamin D3 and signal via vitamin D3 receptor,modulating mammary epithelial cell growth

The vitamin D₃ receptor (VDR) is present in all microenvironments of the breast, yet it is hypothesized to signal through the epithelium to regulate hormone induced growth and differentiation. However, the influence or contribution of the other microenvironments within the breast that express VDR, like the breast adipose tissue, are yet to be investigated. We hypothesized that the breast adipocytes express the signaling components necessary to participate in vitamin D₃ synthesis and signaling via VDR, modulating ductal epithelial cell growth and differentiation. We utilized human primary breast adipocytes and VDR wild type (WT) and knockout (KO) mice to address whether breast adipocytes participate in vitamin D₃‐induced growth regulation of the ductal epithelium. We report in this study that breast primary adipocytes express VDR, CYP27B1 (1α‐hydroxylase, 1α‐OHase), the enzyme that generates the biologically active VDR ligand, 1α,25‐dihydroxyvitamin D₃ (1,25D₃), and CYP24 (24‐hydroxylase, 24‐OHase), a VDR‐1,25D₃ induced target gene. Furthermore, the breast adipocytes participate in bioactivating 25‐hydroxyvitamin D₃ (25D₃) to the active ligand, 1,25D₃, and secreting it to the surrounding microenvironment. In support of this concept, we report that purified mammary ductal epithelial fragments (organoids) from VDR KO mice, co‐cultured with WT breast adipocytes, were growth inhibited upon treatment with 25D₃ or 1,25D₃ compared to vehicle alone. Collectively, these results demonstrate that breast adipocytes bioactivate 25D₃ to 1,25D₃, signal via VDR within the adipocytes, and release an inhibitory factor that regulates ductal epithelial cell growth, suggesting that breast adipose tissue contributes to vitamin D₃‐induced growth regulation of ductal epithelium. J. Cell. Biochem. 112: 3393–3405, 2011. © 2011 Wiley Periodicals, Inc.     

Selection and optimization of MCF‐7 cell line for screening selective inhibitors of 11β‐hydroxysteroid dehydrogenase 2

An 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) produces glucocorticoid (GC) from 11‐keto metabolite, and its modulation has been suggested as a novel approach to treat metabolic diseases. In contrast, type 2 isozyme 11β‐HSD2 is involved in the inactivation of glucocorticoids (GCs), protecting the non‐selective mineralocorticoid receptor (MR) from GCs in kidney. Therefore, when 11β‐HSD1 inhibitors are pursued to treat the metabolic syndrome, preferential selectivity of inhibitors for type 1 over type 2 isozyme is rather important than inhibitory potency. Primarily, to search for cell lines with 11β‐HSD2 activity, we investigated the expression profiles of enzymes or receptors relevant to GC metabolism in breast, colon, and bone‐derived cell lines. We demonstrated that MCF‐7 cells had high expression for 11β‐HSD2, but not for 11β‐HSD1 with its cognate receptor. Next, for the determination of enzyme activity indirectly, we adopted homogeneous time resolved fluorescence (HTRF) cortisol assay. Obviously, the feasibility of HTRF to cellular 11β‐HSD2 was corroborated by constructing inhibitory response to an 11b‐HSD2 inhibitor glycyrrhetinic acid (GA). Taken together, MCF‐7 that overexpresses type 2 but not type 1 enzyme is chosen for cellular 11β‐HSD2 assay, and our results show that a nonradioactive HTRF assay is applicable for type 2 as well as type 1 isozyme. Copyright © 2010 John Wiley & Sons, Ltd.     

Estrogen Reduces 11β‐Hydroxysteroid Dehydrogenase Type 1 in Liver and Visceral,but Not Subcutaneous,Adipose Tissue in Rats

Following menopause, body fat is redistributed from peripheral to central depots. This may be linked to the age related decrease in estrogen levels. We hypothesized that estrogen supplementation could counteract this fat redistribution through tissue‐specific modulation of glucocorticoid exposure. We measured fat depot masses and the expression and activity of the glucocorticoid‐activating enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1) in fat and liver of ovariectomized female rats treated with or without 17β‐estradiol. 11βHSD1 converts inert cortisone, or 11‐dehydrocorticosterone in rats into active cortisol and corticosterone. Estradiol‐treated rats gained less weight and had significantly lower visceral adipose tissue weight than nontreated rats (P < 0.01); subcutaneous adipose weight was unaltered. In addition, 11βHSD1 activity/expression was downregulated in liver and visceral, but not subcutaneous, fat of estradiol‐treated rats (P < 0.001 for both). This downregulation altered the balance of 11βHSD1 expression and activity between adipose tissue depots, with higher levels in subcutaneous than visceral adipose tissue of estradiol‐treated animals (P < 0.05 for both), opposite the pattern in ovariectomized rats not treated with estradiol (P < 0.001 for mRNA expression). Thus, estrogen modulates fat distribution, at least in part, through effects on tissue‐specific glucocorticoid metabolism, suggesting that estrogen replacement therapy could influence obesity related morbidity in postmenopausal women.     

Insulin Action on Expression of Novel Adipose Genes in Healthy and Type 2 Diabetic Subjects

Objective: Adipose tissue secretes several molecules that may participate in metabolic cross‐talk to other insulin‐sensitive tissues. Thus, adipose tissue is a key endocrine organ that regulates insulin sensitivity in other peripheral insulin target tissues. We have studied the expression and acute insulin regulation of novel genes expressed in adipose tissue that are implicated in the control of whole body insulin sensitivity. Research Methods and Procedures: Expression of adiponectin, c‐Cbl—associated protein (CAP), 11‐β hydroxysteroid dehydrogenase type 1 (11β‐HSD‐1), and sterol regulatory element binding protein (SREBP)‐1c was determined in subcutaneous adipose tissue from type 2 diabetic and age‐ and BMI‐matched healthy men by real‐time polymerase chain reaction analysis. Results: Expression of adiponectin, CAP, 11β‐HSD‐1, and SREBP‐1c was similar between healthy and type 2 diabetic subjects. Insulin infusion for 3 hours did not affect expression of CAP, 11β‐HSD‐1, or adiponectin mRNA in either group. However, insulin infusion increased SREBP‐1c expression by 80% in healthy, but not in type 2 diabetic, subjects. Discussion: Our results provide evidence that insulin action on SREBP‐1c is dysregulated in adipose tissue from type 2 diabetic subjects. Impaired insulin regulation on gene expression of select targets in adipose tissue may contribute to the pathogenesis of type 2 diabetes.     

11beta-hydroxysteroid dehydrogenase type 1 in adipose tissue and prospective changes in body weight and insulin resistance

Objective: Increased mRNA and activity levels of 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1) in human adipose tissue (AT) are associated with obesity and insulin resistance. The aim of our study was to investigate whether 11βHSD1 expression or activity in abdominal subcutaneous AT of non‐diabetic subjects are associated with subsequent changes in body weight and insulin resistance [homeostasis model assessment of insulin resistance (HOMA‐IR)]. Research Methods and Procedures: Prospective analyses were performed in 20 subjects (two whites and 18 Pima Indians) who had baseline measurements of 11βHSD1 mRNA and activity in whole AT (follow‐up, 0.3 to 4.9 years) and in 47 Pima Indians who had baseline assessments of 11βHSD1 mRNA in isolated adipocytes (follow‐up, 0.8 to 5.3 years). Results: In whole AT, although 11βHSD1 mRNA levels showed positive associations with changes in weight and HOMA‐IR, 11βHSD1 activity was associated with changes in HOMA‐IR but not in body weight. 11βHSD1 mRNA levels in isolated adipocytes were not associated with follow‐up changes in any of the anthropometric or metabolic variables. Discussion: Our results indicate that increased expression of 11βHSD1 in subcutaneous abdominal AT may contribute to risk of worsening obesity and insulin resistance. This prospective relationship does not seem to be mediated by increased 11βHSD1 expression in adipocytes.     

Luteoprotective roles of luteinizing hormone are mediated by not only progesterone production but also glucocorticoid conversion in bovine corpus luteum

Luteinizing hormone (LH) is known as a key regulator of corpus luteum (CL) function, but the luteoprotective mechanisms of LH in the maintenance of bovine CL function are not well understood. The current study investigated if LH increases cell viability and induces cortisol conversion, and if the luteoprotective action of LH is mediated by stimulating the local production and action of progesterone (P4) and/or cortisol. Cultured bovine luteal cells obtained at the mid‐luteal stage (Days 8–12 of the estrous cycle) were treated for 24 hr with LH (10 ng/ml) with/without onapristone (OP, a specific P4 receptor antagonist; 100 µM), cortisone (1 µM), and aminoglutethimide (AGT, a specific inhibitor of cytochrome P450 side‐chain cleavage; 100 µM). LH with and without OP significantly increased the mRNA and protein expressions of 11β‐hydroxysteroid dehydrogenase (HSD11B) 1, but did not affect the mRNA or protein expression of HSD11B2. These treatments also significantly increased HSD11B1 activity. Cell viability was significantly increased by LH alone or by LH in combination with cortisone and OP. LH in combination with OP or AGT significantly decreased cell viability as compared to LH alone. The overall results suggest that LH stimulates not only P4 production but also HSD11B1 expression, thereby increasing the cortisol concentration in the bovine CL, and that LH prevents cell death through these survival pathways. LH may consequently support CL function during the luteal phase in cattle. Mol. Reprod. Dev. 80: 204–211, 2013. © 2013 Wiley Periodicals, Inc.