beta-HSD Activity Research Articles

Effects of genistein and equol on human and rat testicular 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities

The objective of the present study was to investigate the effects of genistein and equol on 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase 3 (17beta-HSD3) in human and rat testis microsomes. These enzymes (3beta-HSD and 17beta-HSD3), along with two others (cytochrome P450 side-chain cleavage enzyme and cytochrome P450 17alpha-hydroxylase/17-20 lyase), catalyze the reactions that convert the steroid cholesterol into the sex hormone testosterone. Genistein inhibited 3beta-HSD activity (0.2 micromol L(-1) pregnenolone) with half-maximal inhibition or a half-maximal inhibitory concentration (IC(50)) of 87 +/- 15 (human) and 636 +/- 155 nmol L(-1) (rat). Genistein's mode of action on 3beta-HSD activity was competitive for the substrate pregnenolonrge and noncompetitive for the cofactor NAD(+). There was no difference in genistein's potency of 3beta-HSD inhibition between intact rat Leydig cells and testis microsomes. In contrast to its potent inhibition of 3beta-HSD, genistein had lesser effects on human and rat 17beta-HSD3 (0.1 micromol L(-1) androstenedione), with an IC(50) >or= 100 micromol L(-1). On the other hand, equol only inhibited human 3beta-HSD by 42%, and had no effect on 3beta-HSD and 17beta-HSD3 in rat tissues. These observations imply that the ability of soy isoflavones to regulate androgen biosynthesis in Leydig cells is due in part to action on Leydig cell 3beta-HSD activity. Given the increasing intake of soy-based food products and their potential effect on blood androgen levels, these findings are greatly relevant to public health.

Testosterone Stimulates Adipose Tissue 11β-Hydroxysteroid Dehydrogenase Type 1 Expression in a Depot-Specific Manner in Children

Activation of the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in adipose tissue results in the production of excess tissue glucocorticoids and the induction of adiposity and visceral obesity in particular. Androgens may affect body fat distribution by regulating the local metabolism of cortisol. Our objective was to study 11beta-HSD1 mRNA expression in abdominal sc and omental (om) adipose tissue in children after in vitro testosterone and cortisol treatment. Paired fat biopsies (sc and om) were obtained from 19 boys (age 6-14 yr, body mass index 14.6-25.3 kg/m(2), BMI sd score SDS -1.6-3.1) undergoing open abdominal surgery. Pieces of adipose tissue were incubated with testosterone, cortisol, or both hormones for 24 h, whereupon mRNA expression of 11beta-HSD1 and hexose-6-phosphate dehydrogenase (H6PDH) were measured by real-time PCR, and 11beta-HSD1 enzyme activity was determined. Testosterone treatment up-regulated 11beta-HSD1 mRNA expression compared with control incubations in the absence of testosterone (P < 0.05) in om adipose tissue. Testosterone and cortisol both increased 11beta-HSD1 mRNA expression in om but not sc adipose tissue in a depot-specific manner by 2.5- and 2.9-fold, respectively (P < 0.001). However, there was no synergistic effect of the two hormones. 11beta-HSD1 enzyme activity correlated positively to mRNA expression (r = 0.610; P = 0.001). Adipose tissue mRNA expression of H6PDH was affected in a similar fashion to 11beta-HSD1 after hormonal treatment. Testosterone and cortisol stimulated 11beta-HSD1 and H6PDH mRNA expression and 11beta-HSD1 activity in om but not in sc adipose tissue. This suggests that these hormones may contribute to fat distribution and accumulation during childhood.

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 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) in fat and liver of ovariectomized female rats treated with or without 17beta-estradiol. 11betaHSD1 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, 11betaHSD1 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 11betaHSD1 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.

Effects of Proportions of Dietary Macronutrients on Glucocorticoid Metabolism in Diet-Induced Obesity in Rats

Tissue glucocorticoid levels in the liver and adipose tissue are regulated by regeneration of inactive glucocorticoid by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and inactivation by 5α- and 5β-reductases. A low carbohydrate diet increases hepatic 11β-HSD1 and reduces glucocorticoid metabolism during weight loss in obese humans. We hypothesized that similar variations in macronutrient proportions regulate glucocorticoid metabolism in obese rats. Male Lister Hooded rats were fed an obesity-inducing ad libitum ‘Western’ diet (37% fat, n = 36) for 22 weeks, then randomised to continue this diet (n = 12) or to switch to either a low carbohydrate (n = 12) or a moderate carbohydrate (n = 12) diet for the final 8 weeks. A parallel lean control group were fed an ad libitum control diet (10% fat, n = 12) throughout. The low and moderate carbohydrate diets decreased hepatic 11β-HSD1 mRNA compared with the Western diet (both 0.7±0.0 vs 0.9±0.1 AU; p<0.01), but did not alter 11β-HSD1 in adipose tissue. 5α-Reductase mRNA was increased on the low carbohydrate compared with the moderate carbohydrate diet. Compared with lean controls, the Western diet decreased 11β-HSD1 activity (1.6±0.1 vs 2.8±0.1 nmol/mcg protein/hr; p<0.001) and increased 5α-reductase and 5β-reductase mRNAs (1.9±0.3 vs 1.0±0.2 and 1.6±0.1 vs 1.0±0.1 AU respectively; p<0.01) in the liver, and reduced 11β-HSD1 mRNA and activity (both p<0.01) in adipose tissue. Although an obesity-inducing high fat diet in rats recapitulates the abnormal glucocorticoid metabolism associated with human obesity in liver (but not in adipose tissue), a low carbohydrate diet does not increase hepatic 11β-HSD1 in obese rats as occurs in humans.

Tissue-Specific Increases in 11β-Hydroxysteroid Dehydrogenase Type 1 in Normal Weight Postmenopausal Women

With age and menopause there is a shift in adipose distribution from gluteo-femoral to abdominal depots in women. Associated with this redistribution of fat are increased risks of type 2 diabetes and cardiovascular disease. Glucocorticoids influence body composition, and 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) which converts inert cortisone to active cortisol is a putative key mediator of metabolic complications in obesity. Increased 11βHSD1 in adipose tissue may contribute to postmenopausal central obesity. We hypothesized that tissue-specific 11βHSD1 gene expression and activity are up-regulated in the older, postmenopausal women compared to young, premenopausal women. Twenty-three pre- and 23 postmenopausal, healthy, normal weight women were recruited. The participants underwent a urine collection, a subcutaneous adipose tissue biopsy and the hepatic 11βHSD1 activity was estimated by the serum cortisol response after an oral dose of cortisone. Urinary (5α-tetrahydrocortisol+5β-tetrahydrocortisol)/tetrahydrocortisone ratios were higher in postmenopausal women versus premenopausal women in luteal phase (P<0.05), indicating an increased whole-body 11βHSD1 activity. Postmenopausal women had higher 11βHSD1 gene expression in subcutaneous fat (P<0.05). Hepatic first pass conversion of oral cortisone to cortisol was also increased in postmenopausal women versus premenopausal women in follicular phase of the menstrual cycle (P<0.01, at 30 min post cortisone ingestion), suggesting higher hepatic 11βHSD1 activity. In conclusion, our results indicate that postmenopausal normal weight women have increased 11βHSD1 activity in adipose tissue and liver. This may contribute to metabolic dysfunctions with menopause and ageing in women.

Do Alterations in Placental 11β-Hydroxysteroid Dehydrogenase (11βHSD) Activities Explain Differences in Fetal Hypothalamic-Pituitary-Adrenal (HPA) Function Following Periconceptional Undernutrition or Twinning in Sheep?

Periconceptional undernutrition (UN) in sheep accelerates fetal hypothalamic-pituitary-adrenal (HPA) axis activation, resulting in preterm birth. In contrast, twin conception suppresses fetal HPA function and delays prepartum HPA activation. We hypothesized that these dissimilar effects on fetal HPA activity result from different influences of maternal glucocorticoid (GC) on maturation of the fetal HPA axis, mediated via different activities of placental 11beta-hydroxysteroid dehydrogenase (11betaHSD) isozymes. We examined the effects of twinning and maternal periconceptional UN from 60 days before until 30 days after mating on the ontogeny of placental 11betaHSD-1 and -2 enzyme activities. At day 85 of gestation, placental 11betaHSD-2 activity was lower in UN than in normally nourished (N) fetuses (P < .05) and was higher in twins than in singletons (P < .05). Furthermore, placental 11betaHSD-1 activity was not different between nutritional groups but was higher in twins than in singletons (P = .01). At day 85, fetal plasma cortisol (P < .001) and cortisone (P < .001) concentrations were lower in UN than in N fetuses, but the cortisol to cortisone ratio was higher in UN than in N fetuses (P = .01). There was no effect of fetus number on plasma cortisol or cortisone concentrations or on the ratio of cortisol to cortisone at day 85. Therefore, periconceptional UN and twinning may result in the alterations of placental 11betaHSD isozyme activities at particular times during gestation. Changes in these activities during critical periods of fetal development could affect transplacental transfer or placental generation of GCs that reach the fetus, potentially influencing the timing of activation of the fetal HPA axis, fetal maturation, and hence the development and health later in life.

HPLC method for determination of fluorescence derivatives of cortisol, cortisone and their tetrahydro- and allo-tetrahydro-metabolites in biological fluids

11Beta-hydroxysteroid dehydrogenase isoform 2 (11beta-HSD2) is responsible for conversion of cortisol (F) to inactive cortisone (E). Disturbance of its activity can cause hypertension. To estimate 11beta-HSD2 activity, besides F and E, their tetrahydro- (THF, THE) as well allo-tetrahydro- (allo-THF, allo-THE) metabolites should be determined. This study describes HPLC-FLD method for the quantitative determination of endogenous glucocorticoids (GCs) in plasma and urine (total and free) and their metabolites in urine. Following extraction at pH 7.4 using dichloromethane, GCs (F, E, THF, allo-THF, THE, allo-THE and internal standard--prednisolone) were derivatized with 9-anthroyl nitrile and purified by SPE using C(18) cartridges. The enzymatic hydrolysis of conjugated steroids was provided using beta-glucuronidase. The influence of organic bases on 9-AN derivatization of steroids was investigated. The best yield of the derivatization was obtained in presence of the mixture of 10.0% triethylamine (TEA) and 0.1% quinuclidine (Q). Chromatographic separation was accomplished in the Chromolith RP-18e monolithic column. The elaborated method was validated. Calibration curves were linear in the ranges: for F, E and THF 5.0-1000.0 ng mL(-1), for allo-THF and THE + allo-THE 10.0-1000.0 ng mL(-1). LOD (S/N=3:1) for all analytes amounted 3.0 ng mL(-1). Recoveries of GCs exceeded 90%. The method was precise and accurate, intra- and inter-day precision were 3.0-12.1% and 9.2-14.0%, respectively. Accuracy ranged from 0.2 to 15.1%. The method was applied for estimating endogenous GCs in plasma and urine. Plasma levels of F and E were in the ranges: 133.0-174.5 ng mL(-1) and 17.4-35.9 ng mL(-1), respectively. Free urinary steroids were in the ranges: 12.0-54.1 microg/24 h (UFF) and 37.8-76.2 microg/24 h (UFE). The ratio of (THF + allo-THF)/(THE + allo-THE) amounted from 1.01 to 1.23. The obtained results confirmed utility of the elaborated method in the assessment of 11beta-HSD2 activity in man.

Functional effects of polymorphisms in the human gene encoding 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1): a sequence variant at the translation start of 11 beta-HSD1 alters enzyme levels.

Regeneration of active glucocorticoids within liver and adipose tissue by the enzyme 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) may be of pathophysiological importance in obesity and metabolic syndrome and is a therapeutic target in type 2 diabetes. Polymorphisms in HSD11B1, the gene encoding 11 beta-HSD1, have been associated with metabolic phenotype in humans, including type 2 diabetes and hypertension. Here, we have tested the functional consequences of two single nucleotide polymorphisms located in contexts that potentially affect tissue levels of 11 beta-HSD1. We report no effect of allelic variation at rs846910, a polymorphism within the 5'-flanking region of the gene on HSD11B1 promoter activity in vitro. However, compared with the common G allele, the A allele of rs13306421, a polymorphism located two nucleotides 5' to the translation initiation site, gave higher 11 beta-HSD1 expression and activity in vitro and was translated at higher levels in in vitro translation reactions, possibly associated with a lower frequency of "leaky scanning." These data suggest that this polymorphism may have direct functional consequences on levels of 11 beta-HSD1 enzyme activity in vivo. However, the rs13306421 A sequence variant originally reported in other ethnic groups may be of low prevalence because it was not detected in a population of 600 European Caucasian women.

The Role of 11ß-Hydroxysteroid Dehydrogenase 1 in Adipogenesis in Thyroid-Associated Ophthalmopathy

Thyroid-associated ophthalmopathy (TAO) is a sight-threatening autoimmune disease in which de novo adipogenesis has been identified as a fundamental pathogenic mechanism. 11beta-Hydroxysteroid dehydrogenase 1 (11beta-HSD1) increases cortisol bioavailability and is pivotal in mediating glucocorticoid responses in adipose tissue and inflammation. In this study we characterize 11beta-HSD1 as a determinant of the adipogenic and inflammatory pathways in TAO orbital fat (OF) compared with normal OF. OF was harvested from 46 TAO and 44 control patients undergoing orbital surgery. Samples were examined by a combination of immunohistochemistry, real-time RT-PCR, primary cell culture, specific enzyme assays, colorimetric proliferation assays, and bead-based ELISA. Glucocorticoid (glucocorticoid receptor-alpha,11beta-HSD1, hexose-6-phosphate dehydrogenase) and inflammatory cytokines (IL-1beta, IL-1 receptor, IL-6, TNF-alpha, TNF-alpha inductible protein, TGF-beta2) target genes together with markers of late adipocyte differentiation (fatty-acid-binding-protein-4, glycerol-6-phosphate-dehydrogenase) were highly expressed in TAO whole OF (P < 0.05) compared with controls. Primary cultures of TAO OF stromal cells demonstrated greater 11beta-HSD1 oxoreductase activity (P < 0.05), which was regulated by cytokines, most notably TNF-alpha (P < 0.01), compared with controls. Activity increased across differentiation, and this was most marked in TAO cells (P < 0.01). Similarly, stromal cell proliferation was limited by incubation with cortisol in TAO cells only. Furthermore, cortisone decreased IL-6 (P < 0.005), IL-8 (P < 0.05), and macrophage chemoattractant protein-1 (P < 0.05) production by cultured TAO cells only, an effect that was abrogated by inhibition of 11beta-HSD1. Induction of 11beta-HSD1 activity and expression by inflammatory cytokines (TNF-alpha, IL-6) may enhance orbital adipocyte differentiation (adipogenesis) and limit proliferation in TAO. 11beta-HSD1 may also have a role in regulating the local orbital inflammatory response.

Size Distribution of Luteal Cells During Pseudopregnancy in Domestic Cats

Experiments were designed to investigate the size distribution of queen steroidogenic luteal cells throughout pseudopregnancy. Corpora lutea were obtained from the queens following ovariohysterectomy on days 7, 15 or 25 of pseudopregnancy. Luteal cells were isolated from the ovary by collagenase digestion. Steriodogenic cells were identified by staining of cells for 3beta-HSD activity. Cell diameters were measured using a microscope. Luteal cells having steroidogenic capacity covered a wide spectrum of sizes ranging from 3 to 35 mum in diameter. There was a significant increase in mean cell diameters (p < 0.01) as pseudopregnancy progressed. Mean diameter of 3beta-HSD positive cells increased from 10.41 +/- 0.7 microm, on day 7 of pseudopregnancy, to 19.72 +/- 1.3 microm on day 25 of pseudopregnancy. The ratio of large (>20 microm in diameter) to small (3-20 microm in diameter) luteal cells was 0.08 : 1.0 on day 7 of pseudopregnancy, with the 7.5-10 microm cell size class predominant. By day 25 of pseudopregnancy, the ratio of large-to-small cells was increased to 0.87 : 1.0, and 20-25 microm cell sizes become predominant. In conclusion, this study has demonstrated that the cells of the corpus luteum undergo continuous differentiation during pseudopregnancy in queen. This study also demonstrates that luteal cells dissociated from pseudopregnant queen can be used as a model to study the physiology of corpus luteum in pregnant cats.

Novel effects of deoxycorticosterone on testicular 11beta-hydroxysteroid dehydrogenase activity and plasma testosterone levels in normal and adrenalectomized rats.

The 11beta-hydroxysteroid dehydrogenase (11beta-HSD) protects the testis from the inhibitory effects of corticosterone on testosterone (T) production. The objectives of the present studies were to determine the effects of deoxycorticosterone (DOC) and its mechanism of actions on testicular 11beta-HSD activity and plasma T levels after 7 days of treatment. The results revealed that at the end of 7 days treatment, DOC significantly increased testicular 11beta-HSD activity and plasma T levels in normal rats. However, the time course showed that high plasma T levels lowered 11beta-HSD activity on day 14 and by 21 days both the levels normalized. In adrenalectomized (ADX) rats, only the enzyme activity increased significantly but not plasma T levels. Spironolactone, a competitive inhibitor of mineralocorticoid receptor (MR), did not change testicular 11beta-HSD activity in both normal and DOC treated rats suggesting that DOC did not act through MR in increasing 11beta-HSD activity. On the other hand, spironolactone significantly decreased plasma T levels in DOC treated rats. Progesterone (P), a competitive inhibitor of glucocorticoid receptors (GR) or corticosterone significantly suppressed testicular enzyme activity and plasma T levels in DOC treated normal rats. Carbenoxolone which is an inhibitor of 11beta-HSD activity significantly depressed testicular 11beta-HSD activity and plasma T levels in DOC treated normal rats. This paper suggests that DOC increased testicular 11beta-HSD activity through GR; whilst increase in plasma T levels required functioning adrenal glands. The testicular 11beta-HSD is one of the regulators of T levels and vice versa.

Omental Adipose Tissue Type 1 11β-Hydroxysteroid Dehydrogenase Oxoreductase Activity, Body Fat Distribution, and Metabolic Alterations in Women

Modulation of adipose tissue exposure to active glucocorticoids by type 1 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD1) is involved in abdominal obesity of rodent models, but only a few studies have related 11 beta-HSD1 oxoreductase activity to fat distribution in humans. The objective of the study was to examine the link between 11 beta-HSD1 oxoreductase activity, fat distribution patterns, and the metabolic profile in women. Omental (OM) and sc adipose tissue samples were obtained from 36 lean to obese women (aged 47.2 +/- 5.3 yr; body mass index 29.1 +/- 5.2 kg/m(2)) undergoing gynecological surgery. Measures of body composition, fat distribution, blood lipids, and insulin sensitivity were obtained. 11 beta-HSD1 oxoreductase activity was measured over a 24-h period by the reduction of [(14)C]cortisone in adipose tissue homogenates. 11 beta-HSD1 oxoreductase activity was higher in OM compared with sc adipose tissue (9.6 +/- 4.9 vs. 7.9 +/- 4.2 pmol/mg x h, P < 0.01). OM 11 beta-HSD1 oxoreductase activity was positively associated with OM adipocyte size (r = 0.67, P < 0.0001) and visceral adipose tissue area (r = 0.57, P < 0.0003). A positive correlation was also observed between the OM/sc 11 beta-HSD1 oxoreductase activity ratio and the OM/sc adipocyte size ratio (r = 0.37, P < 0.05) as well as the visceral/sc adipose tissue area ratio (r = 0.36, P < 0.05). Women in the highest tertile of OM 11 beta-HSD1 oxoreductase activity had larger OM adipocytes, increased OM lipolysis, increased lipoprotein lipase activity, decreased high-density lipoprotein cholesterol, decreased adiponectin levels, and an increased homeostasis model assessment of insulin resistance index compared with women in the lower tertile (P < 0.05). These results suggest that a relatively higher 11 beta-HSD1 activity in OM vs. sc adipose tissue is associated with preferential visceral fat accumulation and concomitant metabolic alterations.

The p38 Mitogen-Activated Protein Kinase Regulates 11β-Hydroxysteroid Dehydrogenase Type 2 (11β-HSD2) Expression in Human Trophoblast Cells through Modulation of 11β-HSD2 Messenger Ribonucleic Acid Stability

The placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2; encoded by the HSD11B2 gene) has emerged as a key player in controlling fetal development, but its regulation is incompletely understood. Here we identified p38 MAPK as an important regulator of placental 11beta-HSD2. We showed that inhibition of p38 MAPK with the pharmacological inhibitor SB202190 led to an approximately 50% reduction in 11beta-HSD2 activity, protein, and mRNA in primary human placental trophoblast cells. Furthermore, the effect of SB202190 was confirmed by the use of two additional p38 inhibitors, SB203580 and SB220025. In addition, SB202190 decreased the half-life of 11beta-HSD2 mRNA without altering the HSD11B2 promoter activity, indicating that p38 MAPK regulates placental 11beta-HSD2 expression through modulation of 11beta-HSD2 mRNA stability. Importantly, small interfering RNA-mediated knockdown of p38alpha caused a 50% reduction in 11beta-HSD2 activity, suggesting that p38alpha is the primary p38 isoform involved. Taken together, these findings suggest a novel pathway controlling placental 11beta-HSD2 expression resulting from the activation of p38 MAPK. Given that p38alpha is abundantly expressed in the human placenta in which its function is largely unknown, our present study also reveals 11beta-HSD2 as an important target through which p38alpha may regulate human placental function and consequently fetal growth and development.

Placental 11β-HSD2 Gene Expression at Birth Is Inversely Correlated With Growth Velocity in the First Year of Life After Intrauterine Growth Restriction

Intrauterine growth restriction (IUGR) is associated with an increased risk for short stature and diseases in adulthood thought to be inflicted by fetal programming. We hypothesized that placental endocrine systems involved in perinatal growth might also play a role in postnatal growth after IUGR. In a prospective controlled multicenter study, placental gene expression of IGF-binding protein-1 (IGFBP-1), leptin and 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) were measured in 14 IUGR infants and 15 children born appropriate for gestational age (AGA) proven by serial ultrasound examinations. Postnatally, IUGR infants experienced a significantly higher growth velocity than AGA neonates (at 1 y: p = 0.001). Gene expression of 11beta-HSD2 at birth correlated positively with birth length (r = 0.55, p = 0.04) and inversely with growth velocity in the first year of life (r = -0.69, p = 0.01) in the IUGR, but not in the AGA group. There was no correlation between gene expression of placental IGFBP-1, leptin and birth weight, length and growth velocity during the first year of life. AGA infants showed significantly higher concentrations of cortisone in venous cord blood after birth (p = 0.02) as a surrogate of a higher 11beta-HSD2 activity in the fetoplacental unit. In conclusion, placental 11beta-HSD2 gene expression might predict postnatal growth in IUGR.

Antidiabetic effects of 11β‐HSD1 inhibition in a mouse model of combined diabetes, dyslipidaemia and atherosclerosis

11 beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is considered to contribute to the aetiology of the metabolic syndrome, and specific inhibitors have begun to emerge as treatments for insulin resistance and other facets of the syndrome, including atherosclerosis. Given the role of glucocorticoids and 11beta-HSD1 in the anti-inflammatory response and the involvement of inflammation in the development of atherosclerosis, 11beta-HSD1 inhibition may exacerbate atherosclerosis. Our aim was to investigate in vivo the effects of a specific 11beta-HSD1 inhibitor (2922) on atherosclerosis while assessing glucose homeostasis. We conducted a 12-week study administering 2922 (at three doses, 3, 10 and 100 mg/kg body weight) in Ldlr 3KO (Ldlr(-/-)Apob(100/100)Lep(ob/ob)) mice, a genetic model of obesity, insulin resistance, dyslipidaemia and atherosclerosis. Rosiglitazone and simvastatin were used to test the responsiveness of our model in both types of therapy. 2922 was effective in reducing 11beta-HSD1 activity in inguinal adipose tissue (>90% for 100 mg/kg) and was efficacious in improving glucose homeostasis at doses > or =10 mg/kg. Plasma insulin, blood glucose, glucose tolerance and homeostatic model assessment indices were all improved in mice treated with 2922 (100 mg/kg) compared with control animals. Despite an improvement in these parameters, no differences were observed in body weight, adipose or lean tissue masses in the 2922-treated mice. Interestingly, circulating lipids, proinflammatory cytokines and atherosclerosis were unaltered in response to 2922, although a small reduction in LDL cholesterol was detected. Importantly, 11beta-HSD1 inhibition leads to improved glucose metabolism and does not result in a worsening of atherosclerotic lesion area, yet retained antidiabetic potential in the face of multiple severe metabolic aberrations. This study reinforces the potential use of 11beta-HSD1 inhibitors in patients with the metabolic syndrome without negatively impacting atherosclerosis.

Luteinizing hormone induces expression of 11beta-hydroxysteroid dehydrogenase type 2 in rat Leydig cells

BackgroundLeydig cells are the primary source of testosterone in male vertebrates. The biosynthesis of testosterone in Leydig cells is strictly dependent on luteinizing hormone (LH). On the other hand, it can be directly inhibited by excessive glucocorticoid (Corticosterone, CORT, in rats) which is beyond the protective capability of 11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) and type 2 (11beta-HSD2; encoded by gene Hsd11b2 in rats) in Leydig cells. Our previous study found that LH increases 11beta-HSD1 expression in rat Leydig cells, but the effect of LH on the expression and activity of 11beta-HSD2 is not investigated yet.MethodsThe Leydig cells were isolated from male Sprague-Dawley rats (90 days of age). After Leydig cells were incubated either for 24 h with various concentrations of LH (2.5, 5, 10 and 20 ng/mL) or for different time periods (2, 8, 12 and 24 h) with 20 ng/mL LH, the mRNA expression of 11beta-HSD2 was measured by real-time PCR. 11beta-HSD2 protein levels in Leydig cells were assayed by Western Blot and 11beta-HSD2 enzyme activity was determined by calculating the ratio of conversion of [3H]CORT to [3H]11-dehydrocorticosterone by 24 h after stimulation with 20 ng/ml LH. Four reporter gene plasmids containing various lengths of Hsd11b2 promoter region were constructed and transfected into mouse Leydig tumor cells to investigate the effect of LH on Hsd11b2 transcription. A glucocorticoid-responsive reporter gene plasmid, GRE-Luc, was constructed. To evaluate influence of LH on intracellular glucocorticoid level, rat Leydig cells were transfected with GRE-Luc, and luciferase activities were measured after incubation with CORT alone or CORT plus LH.ResultsWe observed dose- and temporal-dependent induction of rat 11beta-HSD2 mRNA expression in Leydig cells subject to LH stimulation. The protein and enzyme activity of 11beta-HSD2 and the luciferase activity of reporter gene driven by promoter regions of Hsd11b2 were increased by LH treatment. LH decreased the glucocorticoid-induced luciferase activity of GRE-Luc reporter gene.ConclusionThe results of the present study suggest that LH increases the expression and enzyme activity of 11beta-HSD2, and therefore enhances capacity for oxidative inactivation of glucocorticoid in rat Leydig cells in vitro.

Regulation of 3 -hydroxysteroid dehydrogenase type 1 and type 2 gene expression and function in the human ovarian surface epithelium by cytokines

The human ovarian surface epithelium (hOSE) is a squamous-to-cuboidal layer that surrounds the ovary. hOSE undergoes injury and repair cycles as a result of ovulation-induced inflammation, an event relevant to the development of epithelial ovarian cancer (EOC). Locally produced steroids mediate the response to inflammation. 3beta-Hydroxysteroid dehydrogenase (3beta-HSD) drives the intracrine generation of progestogens and androgens that potentially affect cell survival and proliferation. We therefore investigated the regulation of 3beta-HSD along with downstream steroid signalling in hOSE. Double immunofluorescence of cultured primary hOSE cells confirmed the expression of 3beta-HSD protein Interleukin (IL). IL-1alpha treatment of primary cells to mimic ovulation-associated inflammation suppressed 3beta-HSD1 expression and stimulated 3beta-HSD2 mRNA (P < 0.001), without affecting total 3beta-HSD protein and activity or androgen or progesterone receptor (PR) mRNA levels. Conversely, IL-4 as a proxy for a post-ovulatory healing cytokine increased both 3beta-HSD transcripts, total protein and activity (P < 0.01). IL-4 also suppressed androgen receptor expression (P < 0.01) without affecting that of the PR, thereby potentially sustaining both progesterone biosynthesis and its underlying signalling in the ovarian surface. 3beta-HSD protein was immunodetectable in primary ascites of women who were diagnosed with EOC but both mRNA transcripts were diminished relative to normal cells (P < 0.05). Notably, this difference was countered by IL-4 treatment (P < 0.01). We conclude that stimulation by IL-4 could be physiologically relevant to post-ovulatory ovarian healing and suggest a novel therapeutic strategy for the activation of progesterone-associated apoptosis in ovarian cancer. Also, our results suggest an attenuation of 3beta-HSD expression in EOC although further studies are required for confirmation.

Effect of continual light deprivation and alpha-2u-globulin replacement therapy on serum concentration of gonadotropins and testicular activity in rats.

Prolonged darkness caused a fall in testicular 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity and diminished spermatogenesis, serum levels of gonadotropins, testosterone and alpha 2u-globulin. Administration of alpha 2u-globulin at a dose of 1.5 mg rat-1 per day for 7 days after 68 days of light deprivation, reversed the 17 beta-HSD activity and serum levels of gonadotropins, testosterone and alpha 2u-globulin, while spermatogenesis was restored to normal. The animals kept in prolonged darkness for 68 days and then received saline (7 days in light-dark cycle, 14 L: 10 D), showed no significant changes of testicular activity, serum levels of gonadotropins, testosterone and alpha 2u-globulin, when compared with dark-exposed animals (68 days) receiving rabbit serum (7 days in light-dark cycle, 14 L: 10 D). These results suggest that alpha 2u-globulin plays an important role in testicular function in dark-exposed rats by inducing gonadotropins and testosterone secretion.

Spermatogenesis in rat: effect of L-tryptophan loading.

The present experiment was designed to assess the role of enhanced central 5-HT level on testicular steroidogenesis and spermatogenesis of rat. L-tryptophan, a precursor in the synthesis of 5-HT in brain, was injected in male rats on a short-term (7-days) and a long-term (21-days) basis. The short-term treatment had no effect on the spermatogenic pattern (Stage - VII of the cycle of the seminiferous epithelium), while the long-term treatment showed a degenerative change (reduced count of the germ cells). Both modes of treatment reduced the activity of testicular delta 5-3 beta-HSD without affecting NADH2-linked Diaphorase activity. Histological study of the Leydig cells' nuclear area also revealed a marked reduction after both the modes of treatment. Human chorionic gonadotropin supplementation in the long-term treatment restored the spermatogenesis, delta 5-3 beta-HSD activity and Leydig cells' nuclear area towards the vehicle treated control. The inhibition of spermatogenesis and steroidogenesis reflects a decrease in the pituitary gonadotropin release under the influence of enhanced 5-HT synthesis in brain, after L-tryptophan loading.

Zinc content in epididymal spermatozoa of metoclopramide-treated rats.

Zinc content was determined separately in spermatozoa taken from epididymal caput and cauda in rats. It was revealed that spermatozoa transported from the epididymal caput to the cauda reduce about 54% of zinc. This reduction is significantly inhibited in spermatozoa of rats receiving metoclopramide. That is also accompanied by a fall of testosterone level in blood serum and of delta 5, 3 beta-HSD activity in Leydig cells. It was found out that the reduction of zinc in spermatozoa at the time of their passage through the epididymis is the process that depends on androgens.