Hexose-6-phosphate dehydrogenase contributes to skeletal muscle homeostasis independent of 11β-hydroxysteroid dehydrogenase type 1

Glucose-6-phosphate (G6P) metabolism by the enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the sarcoplasmic reticulum lumen generates nicotinamide adenine dinucleotide phosphate (reduced) to provide the redox potential for the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to activate glucocorticoid (GC). H6PDH knockout (KO) mice have a switch in 11β-HSD1 activity, resulting in GC inactivation and hypothalamic-pituitary-adrenal axis activation. Importantly, H6PDHKO mice develop a type II fiber myopathy with abnormalities in glucose metabolism and activation of the unfolded protein response (UPR). GCs play important roles in muscle physiology, and therefore, we have examined the importance of 11β-HSD1 and GC metabolism in mediating aspects of the H6PDHKO myopathy. To achieve this, we examined 11β-HSD1/H6PDH double-KO (DKO) mice, in which 11β-HSD1 mediated GC inactivation is negated. In contrast to H6PDHKO mice, DKO mice GC metabolism and hypothalamic-pituitary-adrenal axis set point is similar to that observed in 11β-HSD1KO mice. Critically, in contrast to 11β-HSD1KO mice, DKO mice phenocopy the salient features of the H6PDHKO, displaying reduced body mass, muscle atrophy, and vacuolation of type II fiber-rich muscle, fasting hypoglycemia, increased muscle glycogen deposition, and elevated expression of UPR genes. We propose that muscle G6P metabolism through H6PDH may be as important as changes in the redox environment when considering the mechanism underlying the activation of the UPR and the ensuing myopathy in H6PDHKO and DKO mice. These data are consistent with an 11β-HSD1-independent function for H6PDH in which sarcoplasmic reticulum G6P metabolism and nicotinamide adenine dinucleotide phosphate-(oxidized)/nicotinamide adenine dinucleotide phosphate (reduced) redox status are important for maintaining muscle homeostasis.     

Lack of significant metabolic abnormalities in mice with liver-specific disruption of 11β-hydroxysteroid dehydrogenase type 1

Glucocorticoids (GC) are implicated in the development of metabolic syndrome, and patients with GC excess share many clinical features, such as central obesity and glucose intolerance. In patients with obesity or type 2 diabetes, systemic GC concentrations seem to be invariably normal. Tissue GC concentrations determined by the hypothalamic-pituitary-adrenal (HPA) axis and local cortisol (corticosterone in mice) regeneration from cortisone (11-dehydrocorticosterone in mice) by the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme, principally expressed in the liver. Transgenic mice have demonstrated the importance of 11β-HSD1 in mediating aspects of the metabolic syndrome, as well as HPA axis control. In order to address the primacy of hepatic 11β-HSD1 in regulating metabolism and the HPA axis, we have generated liver-specific 11β-HSD1 knockout (LKO) mice, assessed biomarkers of GC metabolism, and examined responses to high-fat feeding. LKO mice were able to regenerate cortisol from cortisone to 40% of control and had no discernible difference in a urinary metabolite marker of 11β-HSD1 activity. Although circulating corticosterone was unaltered, adrenal size was increased, indicative of chronic HPA stimulation. There was a mild improvement in glucose tolerance but with insulin sensitivity largely unaffected. Adiposity and body weight were unaffected as were aspects of hepatic lipid homeostasis, triglyceride accumulation, and serum lipids. Additionally, no changes in the expression of genes involved in glucose or lipid homeostasis were observed. Liver-specific deletion of 11β-HSD1 reduces corticosterone regeneration and may be important for setting aspects of HPA axis tone, without impacting upon urinary steroid metabolite profile. These discordant data have significant implications for the use of these biomarkers of 11β-HSD1 activity in clinical studies. The paucity of metabolic abnormalities in LKO points to important compensatory effects by HPA activation and to a crucial role of extrahepatic 11β-HSD1 expression, highlighting the contribution of cross talk between GC target tissues in determining metabolic phenotype.     

Relationship of 11β-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase gene polymorphisms with metabolic syndrome and type 2 diabetes

11β-Hydroxysteroid dehydrogenase type 1 (HSD11B1), which converts inactive glucocorticoid to active glucocorticoid, plays a critical role in the pathogenesis of visceral obesity, metabolic syndrome, and diabetes. Hexose-6-phosphate dehydrogenase (H6PD) supplies a crucial cofactor, reduced nicotinamide adenine dinucleotide phosphate (NADPH), which allows HSD11B1 to maintain reductase activity. The association of common SNPs in HSD11B1 [IVS3-29G/T (rs12086634), IVS4-11120A/G (rs1000283)] and H6PD [R453Q (rs6688832), P554L (rs17368528)], either separately or combined, with type 2 diabetes and metabolic syndrome was examined in 427 Korean subjects with type 2 diabetes and in 358 nondiabetic Korean subjects. HSD11B1 polymorphisms (rs12086634 and rs1000283) were associated with metabolic syndrome among type 2 diabetic subjects and an H6PD polymorphism (rs17368528) was a risk factor for metabolic syndrome in nondiabetic subjects. However, no significant association of these SNPs with type 2 diabetes and metabolic syndrome was found after considering the multiple comparisons in the total study population. In conclusion, HSD11B1 and H6PD polymorphisms may not be associated with type 2 diabetes and metabolic syndrome. Further investigation of the role of these gene polymorphisms on the pathogenesis of metabolic syndrome is required.     

Adipose tissue-targeted 11β-hydroxysteroid dehydrogenase type 1 inhibitor protects against diet-induced obesity

Current pharmacological treatments for obesity and metabolic syndrome have various limitations. Recently, adipose tissue 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) has been proposed as a novel therapeutic target for the treatment of obesity and metabolic syndrome. Nevertheless, there is no adipose tissue-targeted 11β-HSD1 inhibitor available now. We sought to develop a new 11β-HSD1 pharmacological inhibitor that homes specifically to the white adipose tissue and aimed to investigate whether adipose tissue-targeted 11β-HSD1 inhibitor might decrease body weight gain and improve glucose tolerance in diet-induced obesity mice. BVT.2733, an 11β-HSD1 selective inhibitor was connected with a peptide CKGGRAKDC that homes to white fat vasculature. CKGGRAKDC-BVT.2733 (T-BVT) or an equimolar mixture of CKGGRAKDC and BVT.2733 (NT-BVT) was given to diet-induced obesity mice for two weeks through subcutaneous injection. T-BVT decreased body weight gain, improved glucose tolerance and decreased adipocyte size compared with vehicle treated mice. In adipose tissue T-BVT administration significantly increased adiponectin, vaspin mRNA levels; In liver T-BVT administration decreased the mRNA level of phosphoenolpyruvate carboxykinase (PEPCK), increased the mRNA levels of mitochondrial carnitine palmi-toyltransferase-I (mCPT-I) and peroxisome proliferator-activated receptorα(PPARα). No significant differences in adipocyte size and hepatic gene expression were observed after treatment with NT-BVT compared with vehicle treated mice, though NT-BVT also decreased body weight gain, improved glucose tolerance, and increased uncoupling protein-2 (UCP-2) mRNA levels in muscle. These results suggest that an adipose tissue-targeted pharmacological inhibitor of 11β-HSD1 may prove to be a new approach for the treatment of obesity and metabolic syndrome.     

Direct regulation of glucose and not insulin on hepatic hexose-6-phosphate dehydrogenase and 11β-hydroxysteroid dehydrogenase type 1

Abnormal hepatic gluconeogenesis contributes significantly to both fasting and non-fasting hyperglycemia of patients with type 2 diabetes. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) regulates the key hepatic gluconeogenic enzymes including phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) through the amplification of glucocorticoid receptor (GR) - mediated tissue glucocorticoid action, and is crucially dependent on hexose-6-phosphate dehydrogenase (H6PDH) - generating NADPH system. Here, we observed that compared with fasting state, H6PDH and 11β-HSD1 expression in livers were all increased under non-fasting state in both normal and diabetic rats, and the non-fasting diabetic group was the highest among the four experimental groups. Moreover, incubation of primary hepatocytes with increasing glucose caused dose-dependent increases in H6PDH, 11β-HSD1, GR, PEPCK and G6Pase expression. Also, glucose-6-phosphate (G6P) had a positive regulation on H6PDH and 11β-HSD1 in hepatocytes. In addition, primary hepatocytes treated with different doses of insulin in high glucose induced alteration of H6PDH and 11β-HSD1 while in low glucose there was no significant effect. These findings suggest that glucose instead of insulin directly regulates H6PDH and 11β-HSD1 and suppression of the two enzymes could be considered as an effective target for the treatment of type 2 diabetes.     

Letrozole ameliorates liver fibrosis through the inhibition of the CTGF pathway and 17β-hydroxysteroid dehydrogenase 13 expression

Journal of Gastroenterology - To establish a treatment option for liver fibrosis, the possibility of the drug repurposing theory was investigated, with a focus on the off-target effects of active...     

Effect of 17 β-estradiol on adherence of escherichia coli to human endometrial stromal cells

Summary The effect of 17 -estradiol onin vitro adherence of six strains of uropathicEscherichia coli to human endometrial stromal cells was examined. In contrast to the findings of earlier investigations using murine urogenital cells and malignant human urogenital cell lines, our studies showed no significant effect of 17 -estradiol on adherence.

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Einflu von 17 -Östradiol auf die Adhärenz von Escherichia coli an menschliche Endometrium-Stromazellen

Zusammenfassung Der Einfluß von 17 -Ostradiol auf dieIn-vitro-Adhärenz von sechs uropathogenenEscherichia-coli-Stämmen an menschliche Endometrium-Stromazellen wurde untersucht. Im Gegensatz zu früheren Untersuchungen, bei denen urogenitale Zellen von Mäusen und maligne urogenitale Zellinien verwendet wurden, zeigten unsere Versuche keinen signifikanten Einfluß des 17 -Östradiol auf die Adhärenz.

     

Estrogen receptor β and 17β-hydroxysteroid dehydrogenase type 6, a growth regulatory pathway that is lost in prostate cancer

Estrogen receptor β (ERβ) is activated in the prostate by 5α-androstane-3β,17β-diol (3β-Adiol) where it exerts antiproliferative activity. The proliferative action of the androgen receptor is activated by 5α-dihydrotestosterone (DHT). Thus, prostate growth is governed by the balance between androgen receptor and ERβ activation. 3β-Adiol is a high-affinity ligand and agonist of ERβ and is derived from DHT by 3-keto reductase/3β-hydroxysteroid dehydrogenase enzymes. Here, we demonstrate that, when it is expressed in living cells containing an estrogen response element-luciferase reporter, 17β-hydroxysteroid dehydrogenase type 6 (17βHSD6) converts the androgen DHT to the estrogen 3β-Adiol, and this leads to activation of the ERβ reporter. This conversion of DHT occurs at concentrations that are in the physiological range of this hormone in the prostate. Immunohistochemical analysis revealed that 17βHSD6 is expressed in ERβ-positive epithelial cells of the human prostate and that, in prostate cancers of Gleason grade higher than 3, both ERβ and 17βHSD6 are undetectable. Both proteins were present in benign prostatic hyperplasia samples. These observations reveal that formation of 3β-Adiol via 17βHSD6 from DHT is an important growth regulatory pathway that is lost in prostate cancer.     

Overexpression of hepatic 5α-reductase and 11β-hydroxysteroid dehydrogenase type 1 in visceral adipose tissue is associated with hyperinsulinemia in morbidly obese patients

11-β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts cortisone to cortisol, mainly in the liver and visceral adipose tissue (VAT), and has been implicated in several metabolic disorders. The absence of systemic hypercortisolism in central obesity could be due to increased inactivation of cortisol to its tetrahydrometabolites by the hepatic enzymes 5α- and 5β-reductases. Our aim was to assess the expression of the reductases in the liver and of 11β-HSD1 in the liver and VAT in morbidly obese patients and to analyze their association with clinical, anthropometric, and biochemical parameters. Hepatic and VAT samples were obtained during bariatric surgery. 5α- and 5β-reductases, 11β-HSD1, and 18S expression was measured using real-time polymerase chain reaction. Anthropometric and biochemical variables were analyzed. Forty-one patients were recruited (age, 41.8 ± 10.6 years; body mass index, 42.1 ± 6.6 kg/m²; 71% women). The expression of hepatic 5α- and 5β-reductases was positively correlated (r = +0.53, P = .004), and their expression levels were correlated with hepatic 11β-HSD1 expression (r = +0.61, P < .001 for 5α-reductase and r = +0.50, P < .001 for 5β-reductase). Hepatic 5α-reductase was associated with insulin (r = +0.34, P = .015). Visceral adipose tissue 11β-HSD1 expression was associated with glucose (r = +0.37, P = .025) and insulin (r = +0.54, P = .002). Our results showed that 5α-reductase and VAT 11β-HSD1 expressions were associated with insulinemia. These findings suggest that overexpression of 5α-reductase, through a higher inactivation of cortisol in the liver, could have a protective role in preserving hepatic sensitivity to insulin. The overexpression of liver reductases in obesity could be an adaptive response to an increase in cortisol production by the liver and visceral 11β-HSD1 to avoid systemic hypercortisolism.     

Expression of vitellogenin in the testis and kidney of the spotted ray Torpedo marmorata exposed to 17β-estradiol

In vertebrates, the liver was long thought to be the only site of vitellogenin (Vtg) production, but recent studies demonstrated that Vtg is also expressed in extrahepatic districts. The aim of this paper is to assess, by in situ hybridization and immunohistochemistry, the expression of Vtg in the testis and kidney of Torpedo marmorata exposed to 17β-estradiol (E₂). In treated samples vtg mRNA and Vtg were detected contemporaneously only in the testis; differently the kidney cells were positive to Vtg antibody, but negative to vtg mRNA. This is the first study to assess that male germ cells, after an exposure to E₂, synthesize Vtg in a stage-dependent manner. The presence of Vtg and the modifications observed in the kidney after E₂ treatment are discussed.     

Discovery of novel sulfonamides as potent and selective inhibitors against human and mouse 11β-hydroxysteroid dehydrogenase type 1

Several classes of non-steroid 11β-HSD1 inhibitors have been developed as promising treatments for Type 2 Diabetes (T2D). Using a human 11β-HSD1 selective inhibitor as a starting point, we designed and synthesized a new class of derivatives of 1-arylsulfonyl piperidine-3-carboxamides. It was found that the large lipophilic group on the amino moiety may lead to cross-species potency towards human and mouse, allowing drug development by evaluating compounds in rodent model. By exploring structure-activity-relationship, the (R)-(+)-bornylamine derivative is identified as the most potent inhibitor of mouse enzyme 11β-HSD1 with an IC(50) of 18 nM. Docking studies revealed the different possible interaction modes of the S-enantiomer and R-enantiomer bound to h11β-HSD1, and explained why the S-enantiomer is more active than the R-enantiomer. Finally, two potent and isoform-selective compounds, (+)-isopinocampheylamine derivative 8m and (R)-(+)-bornylamine derivative 8l, with suitable in vitro properties, could be selected for future PK/PD evaluation in rodent models. Then, 8l was subjected a pharmacodynamics study in vivo with rodent model. It was shown that 8l have 71% and 63% inhibition in adipose and liver tissue at 1h after administration, but it was a short-acting compound displaying a significant drop in potency in the subsequent 3h. This study not only provides compounds as novel h11β-HSD1 inhibitors, but also presents structure-activity relationships for designing potent human/mouse 11β-HSD1 inhibitors suitable for in vivo evaluation in rodent models.     

Methods for long-term 17β-estradiol administration to mice

Rodent models constitute a cornerstone in the elucidation of the effects and biological mechanisms of 17β-estradiol. However, a thorough assessment of the methods for long-term administration of 17β-estradiol to mice is lacking. The fact that 17β-estradiol has been demonstrated to exert different effects depending on dose emphasizes the need for validated administration regimens. Therefore, 169 female C57BL/6 mice were ovariectomized and administered 17β-estradiol using one of the two commonly used subcutaneous methods; slow-release pellets (0.18 mg, 60-day release pellets; 0.72 mg, 90-day release pellets) and silastic capsules (with/without convalescence period, silastic laboratory tubing, inner/outer diameter: 1.575/3.175 mm, filled with a 14 mm column of 36 μg 17β-estradiol/mL sesame oil), or a novel peroral method (56 μg 17β-estradiol/day/kg body weight in the hazelnut cream Nutella). Forty animals were used as ovariectomized and intact controls. Serum samples were obtained weekly for five weeks and 17β-estradiol concentrations were measured using radioimmunoassay. The peroral method resulted in steady concentrations within--except on one occasion--the physiological range and the silastic capsules produced predominantly physiological concentrations, although exceeding the range by maximum a factor three during the first three weeks. The 0.18 mg pellet yielded initial concentrations an order of magnitude higher than the physiological range, which then decreased drastically, and the 0.72 mg pellet produced between 18 and 40 times higher concentrations than the physiological range during the entire experiment. The peroral method and silastic capsules described in this article constitute reliable modes of administration of 17β-estradiol, superior to the widely used commercial pellets.     

Immunolocalization of murine type VI 3β-hydroxysteroid dehydrogenase in the adrenal gland,testis, skin,and placenta

The enzyme 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) is essential for the biosynthesis of all active steroid hormones, such as those secreted from the adrenal gland, testis, ovary, skin and placenta. The 3β-HSD enzymes exist in multiple isoforms in humans and rodents. To date, six different isoforms have been identified in the mouse, and these isoforms are speculated to play different roles in different tissues. We previously showed that the murine type VI 3β-HSD isoform (Hsd3b6) is expressed specifically in the aldosterone-producing zona glomerulosa cells within the adrenal gland and that its overexpression causes abnormally increased aldosterone synthesis, revealing a crucial (or rate-limiting) role of this enzyme in steroidogenesis. However, potential contributions of this enzyme to the steroid hormone synthesis outside the adrenal glands are poorly understood. This paucity of knowledge is partly because of the lack of isoform-specific antibody that can be used for immunohistochemistry. Here, we report the development and characterization of specific antibody to Hsd3b6 and show the results of immunohistochemistry for the adrenal gland, testis, ovary, skin and placenta. As expected, Hsd3b6 immunoreactivities within the adrenal gland were essentially confined to the zona glomerulosa cells, where aldosterone is produced. By contrast, no immunopositive cells were observed in the zona fasciculata, which is where corticosterone is produced. In the gonads, while the ovaries did not show any detectable immunoreactivity to Hsd3b6, the testes displayed intense immunoreactivities within the interstitial Leydig cells, where testosterone is produced. In the skin, positive immunoreactivities to Hsd3b6 were only seen in the sebaceous glands, suggesting a specific role of this enzyme in sebaceous function. Moreover, in the placenta, Hsd3b6 was specifically found in the giant trophoblast cells surrounding the embryonic cavity, which suggests a role for this enzyme in local progesterone production that is required for proper embryonic implantation and/or maintenance of pregnancy. Taken together, our data revealed that Hsd3b6 is localized in multiple specific tissues and cell types, perhaps thereby involved in biosynthesis of a number of tissue-specific steroid hormones with different physiological roles.