Foetal lung maturation in 11beta-hydroxysteroid dehydrogenase type 1 knockout mice.

Glucocorticoids (GCs) induce surfactant synthesis in the late foetal lung. Deficient GC action causes respiratory distress syndrome (RDS). 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts inert cortisone (11-dehydrocorticosterone in rodents) into active cortisol (corticosterone), thus amplifying intracellular GC action. Reduction or loss of pulmonary 11beta-HSD1 activity in glycyrrhetinic acid-treated rats substantially impaired foetal lung maturation (Hundertmark et al., Horm Metab Res, this issue). To test these data, we investigated 11beta-HSD1 activity and lung maturity in the late foetal lung using 11beta-HSD1 knockout mice. Control foetal mice showed high 11beta-HSD activity in the late foetal lung and levels of plasma 11-dehydrocorticosterone were high. Lungs from 11beta-HSD1 -/- mice had lower surfactant protein-A (mRNA and protein) levels and significant depletion of lung surfactant according to both light and electron microscopy, and also had reduced amniotic fluid lecithin/sphingomyelin ratios. These results support the previous experiments with glycyrrhetinic acid and emphasize the importance of 11beta-HSD1 in foetal lung maturation.     

Glucocorticoids, 11 beta-hydroxysteroid dehydrogenase type 1, and visceral obesity

Glucocorticoids are implicated as a pathophysiological mediator of obesity and its accompanying metabolic and cardiovascular complications. Obese patients exhibit normal circulating cortisol levels, related to increased glucocorticoid production and degradation. However, it has been demonstrated that local production of active cortisol from inactive cortisone driven by 11 beta-hydroxysteroid dehydrogenase type 1 is exaggerated in adipose tissue of obese subjects. Such local hypercortisolism may be responsible for increased adipocyte differentiation and enhanced secretion of free fatty acids and other substances involved in the metabolic and cardiovascular complications observed in obesity.     

ABCA1 in adipocytes regulates adipose tissue lipid content,glucose tolerance,and insulin sensitivity

Adipose tissue contains one of the largest reservoirs of cholesterol in the body. Adipocyte dysfunction in obesity is associated with intracellular cholesterol accumulation, and alterations in cholesterol homeostasis have been shown to alter glucose metabolism in cultured adipocytes. ABCA1 plays a major role in cholesterol efflux, suggesting a role for ABCA1 in maintaining cholesterol homeostasis in the adipocyte. However, the impact of adipocyte ABCA1 on adipose tissue function and glucose metabolism is unknown. Our aim was to determine the impact of adipocyte ABCA1 on adipocyte lipid metabolism, body weight, and glucose metabolism in vivo. To address this, we used mice lacking ABCA1 specifically in adipocytes (ABCA1^−ad/−ad). When fed a high-fat, high-cholesterol diet, ABCA1^−ad/−ad mice showed increased cholesterol and triglyceride stores in adipose tissue, developed enlarged fat pads, and had increased body weight. Associated with these phenotypic changes, we observed significant changes in the expression of genes involved in cholesterol and glucose homeostasis, including ldlr, abcg1, glut-4, adiponectin, and leptin. ABCA1^−ad/−ad mice also demonstrated impaired glucose tolerance, lower insulin sensitivity, and decreased insulin secretion. We conclude that ABCA1 in adipocytes influences adipocyte lipid metabolism, body weight, and whole-body glucose homeostasis.     

Hexose-6-phosphate dehydrogenase knock-out mice lack 11 beta-hydroxysteroid dehydrogenase type 1-mediated glucocorticoid generation

The local generation of active glucocorticoid by NADPH-dependent, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) oxoreductase activity, has emerged as an important factor in regulating hepatic glucose output and visceral adiposity. We have proposed that this NADPH is generated within the endoplasmic reticulum by the enzyme hexose-6-phosphate dehydrogenase. To address this hypothesis, we generated mice with a targeted inactivation of the H6PD gene. These mice were unable to convert 11-dehydrocorticosterone (11-DHC) to corticosterone but demonstrated increased corticosterone to 11-DHC conversion consistent with lack of 11beta-HSD1 oxoreductase and a concomitant increase in dehydrogenase activity. This increased corticosterone clearance in the knock-out mice resulted in a reduction in circulating corticosterone levels. Our studies define the critical requirement of hexose-6-phosphate dehydrogenase for 11beta-HSD1 oxoreductase activity and add a new dimension to the investigation of 11beta-HSD1 as a therapeutic target in patients with the metabolic syndrome.     

Piperidine amides as 11beta-hydroxysteroid dehydrogenase type 1 inhibitors

A series of piperidine amide inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) were identified via modifications of the HTS hit compound 1. The synthesis, in vitro biological evaluation, and structure-activity relationship of these compounds are presented.     

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.     

Sub-chronic administration of the 11beta-HSD1 inhibitor, carbenoxolone, improves glucose tolerance and insulin sensitivity in mice with diet-induced obesity

We have examined the metabolic effects of daily administration of carbenoxolone (CBX), a naturally occurring 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) inhibitor, in mice with high fat diet-induced insulin resistance and obesity. Eight-week-old male Swiss TO mice placed on a synthetic high fat diet received daily intraperitoneal injections of either saline vehicle or CBX over a 16-day period. Daily administration of CBX had no effect on food intake, but significantly lowered body weight (1.1- to 1.2-fold) compared to saline-treated controls. Non-fasting plasma glucose levels were significantly decreased (1.6-fold) by CBX treatment on day 4 and remained lower throughout the treatment period. Circulating plasma corticosterone levels were not significantly altered by CBX treatment. Plasma glucose concentrations of CBX-treated mice were significantly reduced (1.4-fold) following an intraperitoneal glucose load compared with saline controls. Similarly, after 16-day treatment with CBX, exogenous insulin evoked a significantly greater reduction in glucose concentrations (1.4- to 1.8-fold). 11beta-HSD1 gene expression was significantly down-regulated in liver, whereas glucocorticoid receptor gene expression was increased in both liver and adipose tissue following CBX treatment. The reduced body weight and improved metabolic control in mice with high fat diet-induced obesity upon daily CBX administration highlights the potential value of selective 11beta-HSD1 inhibition as a new route for the treatment of type 2 diabetes and obesity.     

Oxazolones as potent inhibitors of 11beta-hydroxysteroid dehydrogenase type 1

2,5,5-Trisubstituted oxazolones were identified as potent inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). The synthesis, structure-activity relationship and metabolic stability of these compounds are presented.     

Novel enzymological profiles of human 11beta-hydroxysteroid dehydrogenase type 1

The human enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the reversible oxidoreduction of 11beta-OH/11-oxo groups of glucocorticoid hormones. Besides this important endocrinological property, the type 1 isozyme (11beta-HSD1) mediates reductive phase I reactions of several carbonyl group bearing xenobiotics, including drugs, insecticides and carcinogens. The aim of this study was to explore novel substrate specificities of human 11beta-HSD1, using heterologously expressed protein in the yeast system Pichia pastoris. In addition to established phase I xenobiotic substrates, it is now demonstrated that transformed yeast strains catalyze the reduction of ketoprofen to its hydroxy metabolite, and the oxidation of the prodrug DFU-lactol to the pharmacologically active lactone compound. Purified recombinant 11beta-HSD1 mediated oxidative reactions, however, the labile reductive activity component could not be maintained. In conclusion, evidence is provided that human 11beta-HSD1 in vitro is involved in phase I reactions of anti-inflammatory non-steroidal drugs like ketoprofen and DFU-lactol.     

Novel non-steroidal inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) regulates glucocorticoid action at the pre-receptor stage by converting cortisone to cortisol. 11β-HSD1 is selectively expressed in many tissues including the liver and adipose tissue where metabolic events are important. Metabolic syndrome relates to a number of metabolic abnormalities and currently has a prevalence of >20% in adult Americans. 11β-HSD1 inhibitors are being investigated by many major pharmaceutical companies for type 2 diabetes and other abnormalities associated with metabolic syndrome. In this area of intense interest a number of structural types of 11β-HSD1 inhibitor have been identified. It is important to have an array of structural types as the physicochemical properties of the compounds will determine tissue distribution, HPA effects, and ultimately clinical utility. Here we report the discovery and synthesis of three structurally different series of novel 11β-HSD1 inhibitors that inhibit human 11β-HSD1 in the low micromolar range. Docking studies with 1–3 into the crystal structure of human 11β-HSD1 reveal how the molecules may interact with the enzyme and cofactor and give further scope for structure based drug design in the optimisation of these series.     

PPARalpha agonists reduce 11beta-hydroxysteroid dehydrogenase type 1 in the liver

11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) is an enzyme that converts cortisone to the active glucocorticoid, cortisol. Cortisol-cortisone interconversion plays a key role in the regulation of glucose metabolism, since mice deficient in 11betaHSD1 are resistant to diet-induced hyperglycemia. Peroxisome proliferator activator receptors (PPAR) are key regulators of glucose and lipid homeostasis. We observed a striking downregulation of murine hepatic 11betaHSD1 expression and activity after chronic treatment of wild-type mice with PPARalpha agonists, while 11betaHSD1 in the livers of PPARalpha knockout mice, or in mice treated for only 7 h with PPARalpha agonists, was unaltered. Our results are the first to show PPARalpha agonists can affect glucocorticoid metabolism in the liver by altering 11betaHSD1 expression after chronic treatment. Regulation of active glucocorticoid levels in the liver by PPARalpha agonists may in turn affect glucose metabolism, consistent with reports of their antidiabetic effects.     

Effects of acetazolamide on insulin release, serum glucose and insulin, glucose tolerance, and alloxan sensitivity of mice

Isolated pancreatic islets exposed to 100 mM acetazolamide (AZM) and low glucose concentration exhibited increased insulin release, whereas those subjected to AZM and high glucose concentration exhibited decreased secretion of insulin. A slight transient hyperglycaemia was found 24 h after administration of 1.5 g/kg b.wt. of AZM to fed mice, whereas no such response was seen in starved mice. The serum insulin concentration was increased in the 24 h after AZM injection. Pretreatment with AZM caused decreased glucose tolerance and protection against alloxan toxicity. Inhibited carbonic anhydrase activity and ionic alterations might have played a role in the development of these effects of AZM in mice.     

11beta-hydroxysteroid dehydrogenase 2 activity is elevated in severe obesity and negatively associated with insulin sensitivity

Alterations in glucocorticoid (GC) metabolism may contribute to the development of obesity and insulin resistance. We aimed to study the role of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) in human adiposity, paying special attention to the association between altered GC metabolism and insulin sensitivity. In 24-h urine samples of 72 extremely obese (mean BMI 45.5 +/- 1.1 kg/m(2)), but otherwise healthy patients urinary free cortisol (UFF), urinary free cortisone (UFE), tetrahydrocortisol (THF), 5alpha-tetrahydrocortisol (5alpha-THF), and tetrahydrocortisone (THE) were quantified by radioimmunoassay. The sum of the three major tetrahydrometabolites is an estimate for daily GC secretion, and the sum of UFF and UFE represents potentially bioactive-free-GCs. Thirty healthy lean subjects (BMI 22.3 +/- 0.3 kg/m(2)) served as controls. In obese subjects, absolute daily GC secretion and the potentially bioactive-free-GCs were significantly (P < 0.005) higher than in lean controls (11.8 +/- 0.7 vs. 8.0 +/- 0.6 mg/d; and 171.8 +/- 11.2 vs. 117.6 +/- 9.2 mug/d, respectively). However, when these values were corrected for body surface area (BSA), significant differences were no longer detectable. While enzyme activity indices for 5alpha-reductase and 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) were similar in lean and obese subjects, 11beta-HSD2 was markedly elevated in adiposity (3.7 +/- 0.2 vs. 2.1 +/- 0.1; P < 0.0001). This increase was accompanied by a significant reduction in UFF excretion corrected for BSA (16.5 +/- 1.2 vs. 21.7 +/- 2.0 mug/d/m(2); P = 0.0222). Besides, 11beta-HSD2 activity was significantly correlated with insulin sensitivity (P = 0.0262). When body size is accounted for, both adrenal GC secretion and potentially bioactive-free-GCs are indistinguishable between lean and extremely obese subjects. However in obesity, the kidney appears to intensify its supply of the direct substrate cortisone for extrarenal 11beta-HSD1, which may fuel visceral adiposity and insulin resistance.     

MRNA and enzyme activity of hepatic 11beta-hydroxysteroid dehydrogenase type 1 are elevated in C57BL/KsJ-db/db mice.

To evaluate the importance of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in insulin resistant diabetic C57BL/KsJ-db/db mice, we measured the activity and mRNA level of 11beta-HSD1 in the liver of db/db mice and their heterozygote litter mates, db/+m mice. The blood glucose, plasma insulin, and corticosterone levels of db/db mice were significantly higher than those of db/+m mice. Despite hyperinsulinemia, the activity level of this enzyme was significantly higher in db/db mice, and the mRNA level of hepatic 11beta-HSD1 was also significantly higher in db/db mice. Since hepatic 11beta-HSD1 in vivo mainly functions as 11-keto-reductase and does not work as 11beta-oxidase, these results suggest that the rate of hepatic conversion of 11-dehydrocorticosterone to corticosterone is increased in db/db mice, resulting in higher glucocorticoid activity in the liver. The increased hepatic corticosterone concentration due to the elevation of 11beta-HSD1 and high plasma corticosterone concentration may antagonize the action of insulin and cause insulin resistance. These findings have a potentially important implication for relationships between increased hepatic 11beta-HSD1 and insulin resistance in db/db mice. The present paper is the first to demonstrate the increased activities and mRNA level of hepatic 11beta-HSD1 in db/db mice.     

Long-term administration of maxadilan improves glucose tolerance and insulin sensitivity in mice

Maxadilan and its truncated variant, M65, are agonist and antagonist specific, respectively, for the PAC1 receptor. PAC1 is the specific receptor for the pituitary adenylate cyclase-activating peptide (PACAP), which is not shared by vasoactive intestinal peptide (VIP). PACAP is a ubiquitous peptide of the glucagon superfamily that is involved in glucose homeostasis and regulation of insulin secretion. This study employed the recombinant maxadilan and M65 to evaluate the PAC1 receptor-mediated effects on energy metabolism using NIH mice. First, the acute effect of maxadilan-induced hyperglycemia was blocked by M65. In long-term studies, NIH mice were given daily intraperitoneal injections with maxadilan, M65, or vehicle for 21 days. Maxadilan suppressed feeding and enhanced water intake significantly for the first several days. After that period, maxadilan treatment continued to promote food and water intake. Long-term administration of maxadilan led to an increase in body weight (P<0.01), decrease in body fat (P<0.01), down-regulation of basal plasma glucose (P<0.01), upregulation of basal plasma insulin (P<0.01) and improved glucose tolerance (P<0.01) and insulin sensitivity (P<0.01). An elevation in plasma LDL (P<0.01) was also observed in the maxadilan group. However, M65 displayed no significant adverse effects on the aforementioned parameters except basal plasma glucose (P<0.05). The significant changes induced by maxadilan indicate that the PAC1 receptor plays multiple key roles in carbohydrate metabolism, lipid metabolism and energy homeostasis in mice.     

Bis-aryl triazoles as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1

3-Aryl-5-phenyl-(1,2,4)-triazoles were identified as selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). They are active in both in vitro and an in vivo mouse pharmacodynamic (PD) model. The synthesis and structure activity relationships are presented.     

Adamantyl triazoles as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1

Adamantyl triazoles were identified as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). They are active both in in vitro and in in vivo pharmacodynamic models. The synthesis and structure-activity relationships of these inhibitors are presented.     

Cloning of chicken 11beta-hydroxysteroid dehydrogenase type 1 and its tissue distribution

11beta-Hydroxysteroid dehydrogenase type 1 (11HSD1) is an enzyme that interconverts active 11-hydroxy glucocorticoids (cortisol, corticosterone) and their inactive 11-oxo derivatives (cortisone, 11-dehydrocorticosterone). Although bidirectional, it is considered to operate in vivo as an 11-reductase that regenerates active glucocorticoids and thus amplifies their local activity in mammals. Here we report the cloning, characterization and tissue distribution of chicken 11HSD1 (ch11HSD1). Its cDNA predicts a protein of 300 amino acids that share 51-56% sequence identity with known mammalian 11HSD1 proteins, while in contrast to most mammals, ch11HSD1 contains only one N-linked glycosylation site. Analysis of the tissue distribution pattern by RT-PCR revealed that ch11HSD1 is expressed in a large variety of tissues, with high expression in the liver, kidney and intestine, and weak in the gonads, brain and heart. 11-Reductase activity has been found in the liver, kidney, intestine and gonads with low or almost zero activity in the brain and heart. These results provide evidence for a role of 11HSD1 as a tissue-specific regulator of glucocorticoid action in non-mammalian vertebrates and may serve as a suitable model for further analysis of 11HSD1 evolution in vertebrates.     

High-level production and optimization of monodispersity of 11beta-hydroxysteroid dehydrogenase type 1

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is an intraluminally oriented, endoplasmic reticulum (ER)-bound enzyme catalyzing the interconversion between inactive cortisone and hormonally active cortisol. Heterologous production of 11beta-HSD1, devoid of its N-terminal transmembrane segment, is possible but yields only small amounts of soluble protein. Here we show that the soluble portion of recombinant 11beta-HSD1 produced in E. coli is found mainly as multimeric aggregates in the absence of detergent, and to a large extent associated with the endogenous chaperonin GroEL and other E. coli proteins. By co-overexpressing GroEL/ES and adding an 11beta-HSD1 inhibitor during protein synthesis, we have increased the accumulation of soluble 11beta-HSD1 by more than one order of magnitude. Using monodispersity as a screening criterion, we have also optimized the purification process by evaluating various solubilizing systems for the chromatographic steps, finally obtaining stable monodisperse preparations of both human and guinea pig 11beta-HSD1. By analytical ultracentrifugation, we could demonstrate that 11beta-HSD1 mainly exists as a dimer in the solubilized state. Moreover, active site titration of human 11beta-HSD1 revealed that at least 75% of the protein in a typical preparation represents active enzyme. Equilibrium unfolding experiments indicate that addition of inhibitor and the cofactor NADP(H) can stabilize the conformational stability of this enzyme in an additive manner. The outlined procedure may provide a general method for preparing similar proteins to oligomeric homogeneity and with retained biological activity.