The effect of oral butyrate on colonic short-chain fatty acid transporters and receptors depends on microbial status.

Butyrate, a metabolite produced by gut bacteria, has demonstrated beneficial effects in the colon and has been used to treat inflammatory bowel diseases. However, the mechanism by which butyrate operates remains incompletely understood. Given that oral butyrate can exert either a direct impact on the gut mucosa or an indirect influence through its interaction with the gut microbiome, this study aimed to investigate three key aspects: (1) whether oral intake of butyrate modulates the expression of genes encoding short-chain fatty acid (SCFA) transporters (Slc16a1, Slc16a3, Slc16a4, Slc5a8, Abcg2) and receptors (Hcar2, Ffar2, Ffar3, Olfr78, Olfr558) in the colon, (2) the potential involvement of gut microbiota in this modulation, and (3) the impact of oral butyrate on the expression of colonic SCFA transporters and receptors during colonic inflammation. Specific pathogen-free (SPF) and germ-free (GF) mice with or without DSS-induced inflammation were provided with either water or a 0.5% sodium butyrate solution. The findings revealed that butyrate decreased the expression of Slc16a1, Slc5a8, and Hcar2 in SPF but not in GF mice, while it increased the expression of Slc16a3 in GF and the efflux pump Abcg2 in both GF and SPF animals. Moreover, the presence of microbiota was associated with the upregulation of Hcar2, Ffar2, and Ffar3 expression and the downregulation of Slc16a3. Interestingly, the challenge with DSS did not alter the expression of SCFA transporters, regardless of the presence or absence of microbiota, and the effect of butyrate on the transporter expression in SPF mice remained unaffected by DSS. The expression of SCFA receptors was only partially affected by DSS. Our results indicate that (1) consuming a relatively low concentration of butyrate can influence the expression of colonic SCFA transporters and receptors, with their expression being modulated by the gut microbiota, (2) the effect of butyrate does not appear to result from direct substrate-induced regulation but rather reflects an indirect effect associated with the gut microbiome, and (3) acute colon inflammation does not lead to significant changes in the transcriptional regulation of most SCFA transporters and receptors, with the effect of butyrate in the inflamed colon remaining intact.

Microbiota modulates the steroid response to acute immune stress in male mice.

Microbiota plays a role in shaping the HPA-axis response to psychological stressors. To examine the role of microbiota in response to acute immune stressor, we stimulated the adaptive immune system by anti-CD3 antibody injection and investigated the expression of adrenal steroidogenic enzymes and profiling of plasma corticosteroids and their metabolites in specific pathogen-free (SPF) and germ-free (GF) mice. Using UHPLC-MS/MS, we showed that 4 hours after immune challenge the plasma levels of pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone (CORT), 11-dehydroCORT and their 3α/ß-, 5α-, and 20α-reduced metabolites were increased in SPF mice, but in their GF counterparts, only CORT was increased. Neither immune stress nor microbiota changed the mRNA and protein levels of enzymes of adrenal steroidogenesis. In contrast, immune stress resulted in downregulated expression of steroidogenic genes (Star, Cyp11a1, Hsd3b1, Hsd3b6) and upregulated expression of genes of the 3α-hydroxysteroid oxidoreductase pathway (Akr1c21, Dhrs9) in the testes of SPF mice. In the liver, immune stress downregulated the expression of genes encoding enzymes with 3ß-hydroxysteroid dehydrogenase (HSD) (Hsd3b2, Hsd3b3, Hsd3b4, Hsd3b5), 3α-HSD (Akr1c14), 20α-HSD (Akr1c6, Hsd17b1, Hsd17b2) and 5α-reductase (Srd5a1) activities, except for Dhrs9, which was upregulated. In the colon, microbiota downregulated Cyp11a1 and modulated the response of Hsd11b1 and Hsd11b2 expression to immune stress. These data underline the role of microbiota in shaping the response to immune stressor. Microbiota modulates the stress-induced increase in C21 steroids, including those that are neuroactive that could play a role in alteration of HPA axis response to stress in GF animals.

The ratio of ursodeoxycholyltaurine to 7-oxolithocholyltaurine serves as a biomarker of decreased 11ß-hydroxysteroid dehydrogenase 1 activity in mouse.

BACKGROUND AND PURPOSE: 11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) regulates tissue-specific glucocorticoid metabolism and its impaired expression and activity are associated with major diseases. Pharmacological inhibition of 11ß-HSD1 is considered a promising therapeutic strategy. This study investigated whether alternative 7-oxo bile acid substrates of 11ß-HSD1 or the ratios to their 7-hydroxy products can serve as biomarkers for decreased enzymatic activity. EXPERIMENTAL APPROACH: Bile acid profiles were measured by ultra-HPLC tandem-MS in plasma and liver tissue samples of four different mouse models with decreased 11ß-HSD1 activity: global (11KO) and liver-specific 11ß-HSD1 knockout mice (11LKO), mice lacking hexose-6-phosphate dehydrogenase (H6pdKO) that provides cofactor NADPH for 11ß-HSD1 and mice treated with the pharmacological inhibitor carbenoxolone. Additionally, 11ß-HSD1 expression and activity were assessed in H6pdKO- and carbenoxolone-treated mice. KEY RESULTS: The enzyme product to substrate ratios were more reliable markers of 11ß-HSD1 activity than absolute levels due to large inter-individual variations in bile acid concentrations. The ratio of the 7ß-hydroxylated ursodeoxycholyltaurine (UDC-Tau) to 7-oxolithocholyltaurine (7oxoLC-Tau) was diminished in plasma and liver tissue of all four mouse models and decreased in H6pdKO- and carbenoxolone-treated mice with moderately reduced 11ß-HSD1 activity. The persistence of 11ß-HSD1 oxoreduction activity in the face of H6PD loss indicates the existence of an alternative NADPH source in the endoplasmic reticulum. CONCLUSIONS AND IMPLICATIONS: The plasma UDC-Tau/7oxo-LC-Tau ratio detects decreased 11ß-HSD1 oxoreduction activity in different mouse models. This ratio may be a useful biomarker of decreased 11ß-HSD1 activity in pathophysiological situations or upon pharmacological inhibition. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

Deletion of the serine protease CAP2/Tmprss4 leads to dysregulated renal water handling upon dietary potassium depletion.

The kidney needs to adapt daily to variable dietary K+ contents via various mechanisms including diuretic, acid-base and hormonal changes that are still not fully understood. In this study, we demonstrate that following a K+-deficient diet in wildtype mice, the serine protease CAP2/Tmprss4 is upregulated in connecting tubule and cortical collecting duct and also localizes to the medulla and transitional epithelium of the papilla and minor calyx. Male CAP2/Tmprss4 knockout mice display altered water handling and urine osmolality, enhanced vasopressin response leading to upregulated adenylate cyclase 6 expression and cAMP overproduction, and subsequently greater aquaporin 2 (AQP2) and Na+-K+-2Cl- cotransporter 2 (NKCC2) expression following K+-deficient diet. Urinary acidification coincides with significantly increased H+,K+-ATPase type 2 (HKA2) mRNA and protein expression, and decreased calcium and phosphate excretion. This is accompanied by increased glucocorticoid receptor (GR) protein levels and reduced 11ß-hydroxysteroid dehydrogenase 2 activity in knockout mice. Strikingly, genetic nephron-specific deletion of GR leads to the mirrored phenotype of CAP2/Tmprss4 knockouts, including increased water intake and urine output, urinary alkalinisation, downregulation of HKA2, AQP2 and NKCC2. Collectively, our data unveil a novel role of the serine protease CAP2/Tmprss4 and GR on renal water handling upon dietary K+ depletion.

Interactions Between Gut Microbiota and Acute Restraint Stress in Peripheral Structures of the Hypothalamic-Pituitary-Adrenal Axis and the Intestine of Male Mice.

The gut microbiota play an important role in shaping brain functions and behavior, including the activity of the hypothalamus-pituitary-adrenocortical (HPA) axis. However, little is known about the effect of the microbiota on the distinct structures (hypothalamus, pituitary, and adrenals) of the HPA axis. In the present study, we analyzed the influence of the microbiota on acute restraint stress (ARS) response in the pituitary, adrenal gland, and intestine, an organ of extra-adrenal glucocorticoid synthesis. Using specific pathogen-free (SPF) and germ-free (GF) male BALB/c mice, we showed that the plasma corticosterone response to ARS was higher in GF than in SPF mice. In the pituitary, stress downregulated the expression of the gene encoding CRH receptor type 1 (Crhr1), upregulated the expression of the Fkbp5 gene regulating glucocorticoid receptor sensitivity and did not affect the expression of the proopiomelanocortin (Pomc) and glucocorticoid receptor (Gr) genes. In contrast, the microbiota downregulated the expression of pituitary Pomc and Crhr1 but had no effect on Fkbp5 and Gr. In the adrenals, the steroidogenic pathway was strongly stimulated by ARS at the level of the steroidogenic transcriptional regulator Sf-1, cholesterol transporter Star and Cyp11a1, the first enzyme of steroidogenic pathway. In contrast, the effect of the microbiota was significantly detected at the level of genes encoding steroidogenic enzymes but not at the level of Sf-1 and Star. Unlike adrenal Sf-1, the expression of the gene Lrh-1, which encodes the crucial transcriptional regulator of intestinal steroidogenesis, was modulated by the microbiota and ARS and this effect differed between the ileum and colon. The findings demonstrate that gut microbiota have an impact on the response of the pituitary, adrenals and intestine to ARS and that the interaction between stress and the microbiota during activation of glucocorticoid steroidogenesis differs between organs. The results suggest that downregulated expression of pituitary Pomc and Crhr1 in SPF animals might be an important factor in the exaggerated HPA response of GF mice to stress.

Lack of Renal Tubular Glucocorticoid Receptor Decreases the Thiazide-Sensitive Na+/Cl- Cotransporter NCC and Transiently Affects Sodium Handling.

Chronic glucocorticoid infusion impairs NCC activity and induces a non-dipping profile in mice, suggesting that glucocorticoids are essential for daily blood pressure variations. In this paper, we studied mice lacking the renal tubular glucocorticoid receptor (GR) in adulthood (GR knockouts, Nr3c1 Pax8/LC1 ). Upon standard salt diet, Nr3c1 Pax8/LC1 mice grow normally, but show reduced NCC activity despite normal plasma aldosterone levels. Following diet switch to low sodium, Nr3c1 Pax8/LC1 mice exhibit a transient but significant reduction in the activity of NCC and expression of NHE3 and NKCC2 accompanied by significant increased Spak activity. This is followed by transiently increased urinary sodium excretion and higher plasma aldosterone concentrations. Plasma corticosterone levels and 11ßHSD2 mRNA expression and activity in the whole kidney remain unchanged. High salt diet does not affect whole body Na+ and/or K+ balance and NCC activity is not reduced, but leads to a significant increase in diastolic blood pressure dipping in Nr3c1 Pax8/LC1 mice. When high sodium treatment is followed by 48 h of darkness, NCC abundance is reduced in knockout mice although activity is not different. Our data show that upon Na+ restriction renal tubular GR-deficiency transiently affects Na+ handling and transport pathways. Overall, upon standard, low Na+ and high Na+ diet exposure Na+ and K+ balance is maintained as evidenced by normal plasma and urinary Na+ and K+ and aldosterone concentrations.

Absence of hexose-6-phosphate dehydrogenase results in reduced overall glucose consumption but does not prevent 11ß-hydroxysteroid dehydrogenase-1-dependent glucocorticoid activation.

Hexose-6-phosphate dehydrogenase (H6PD) is thought to be the major source of NADPH within the endoplasmic reticulum (ER), determining 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) reaction direction to convert inert 11-oxo- to potent 11ß-hydroxyglucocorticoids. Here, we tested the hypothesis whether H6pd knock-out (KO) in primary murine bone marrow-derived macrophages results in a switch from 11ß-HSD1 oxoreduction to dehydrogenation, thereby inactivating glucocorticoids (GC) and affecting macrophage phenotypic activation as well as causing a more aggressive M1 macrophage phenotype. H6pdKO did not lead to major disturbances of macrophage activation state, although a slightly more pronounced M1 phenotype was observed with enhanced proinflammatory cytokine release, an effect explained by the decreased 11ß-HSD1-dependent GC activation. Unexpectedly, ablation of H6pd did not switch 11ß-HSD1 reaction direction. A moderately decreased 11ß-HSD1 oxoreduction activity by 40-50% was observed in H6pdKO M1 macrophages but dehydrogenation activity was undetectable, providing strong evidence for the existence of an alternative source of NADPH in the ER. H6pdKO M1 activated macrophages showed decreased phagocytic activity, most likely a result of the reduced 11ß-HSD1-dependent GC activation. Other general macrophage functions reported to be influenced by GC, such as nitrite production and cholesterol efflux, were altered negligibly or not at all. Importantly, assessment of energy metabolism using an extracellular flux analyzer and lactate measurements revealed reduced overall glucose consumption in H6pdKO M1 activated macrophages, an effect that was GC independent. The GC-independent influence of H6PD on energy metabolism and the characterization of the alternative source of NADPH in the ER warrant further investigations. ENZYMES: 11ß-HSD1, EC 1.1.1.146; H6PD, EC 1.1.1.47.

Inhibition of 11ß-hydroxysteroid dehydrogenase 2 by the fungicides itraconazole and posaconazole.

Impaired 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2)-dependent cortisol inactivation can lead to electrolyte dysbalance, hypertension and cardiometabolic disease. Furthermore, placental 11ß-HSD2 essentially protects the fetus from high maternal glucocorticoid levels, and its impaired function has been associated with altered fetal growth and a higher risk for cardio-metabolic diseases in later life. Despite its important role, 11ß-HSD2 is not included in current off-target screening approaches. To identify potential 11ß-HSD inhibitors among approved drugs, a pharmacophore model was used for virtual screening, followed by biological assessment of selected hits. This led to the identification of several azole fungicides as 11ß-HSD inhibitors, showing a significant structure-activity relationship between azole scaffold size, 11ß-HSD enzyme selectivity and inhibitory potency. A hydrophobic linker connecting the azole ring to the other, more polar end of the molecule was observed to be favorable for 11ß-HSD2 inhibition and selectivity over 11ß-HSD1. The most potent 11ß-HSD2 inhibition, using cell lysates expressing recombinant human 11ß-HSD2, was obtained for itraconazole (IC50 139±14nM), its active metabolite hydroxyitraconazole (IC50 223±31nM) and posaconazole (IC50 460±98nM). Interestingly, experiments with mouse and rat kidney homogenates showed considerably lower inhibitory activity of these compounds towards 11ß-HSD2, indicating important species-specific differences. Thus, 11ß-HSD2 inhibition by these compounds is likely to be overlooked in preclinical rodent studies. Inhibition of placental 11ß-HSD2 by these compounds, in addition to the known inhibition of cytochrome P450 enzymes and P-glycoprotein efflux transport, might contribute to elevated local cortisol levels, thereby affecting fetal programming.

Adult nephron-specific MR-deficient mice develop a severe renal PHA-1 phenotype.

Aldosterone is the main mineralocorticoid hormone controlling sodium balance, fluid homeostasis, and blood pressure by regulating sodium reabsorption in the aldosterone-sensitive distal nephron (ASDN). Germline loss-of-function mutations of the mineralocorticoid receptor (MR) in humans and in mice lead to the "renal" form of type 1 pseudohypoaldosteronism (PHA-1), a case of aldosterone resistance characterized by salt wasting, dehydration, failure to thrive, hyperkalemia, and metabolic acidosis. To investigate the importance of MR in adult epithelial cells, we generated nephron-specific MR knockout mice (MR(Pax8/LC1)) using a doxycycline-inducible system. Under standard diet, MR(Pax8/LC1) mice exhibit inability to gain weight and significant weight loss compared to control mice. Interestingly, despite failure to thrive, MR(Pax8/LC1) mice survive but develop a severe PHA-1 phenotype with higher urinary Na(+) levels, decreased plasma Na(+), hyperkalemia, and higher levels of plasma aldosterone. This phenotype further worsens and becomes lethal under a sodium-deficient diet. Na(+)/Cl(-) co-transporter (NCC) protein expression and its phosphorylated form are downregulated in the MR(Pax8/LC1) knockouts, as well as the αENaC protein expression level, whereas the expression of glucocorticoid receptor (GR) is increased. A diet rich in Na(+) and low in K(+) does not restore plasma aldosterone to control levels but is sufficient to restore body weight, plasma, and urinary electrolytes. In conclusion, MR deletion along the nephron fully recapitulates the features of severe human PHA-1. ENaC protein expression is dependent on MR activity. Suppression of NCC under hyperkalemia predominates in a hypovolemic state.

Differential impact of stress on hypothalamic-pituitary-adrenal axis: gene expression changes in Lewis and Fisher rats.

The aim of the present work was to study the influence of variable stress on the expression of 11ß-hydroxysteroid dehydrogenase type 1 (11HSD1) and the neuropeptides corticotropin-releasing hormone (CRH), urocortins 2 and 3(UCN2, UCN3), arginine vasopressin (AVP), oxytocin (OXT) and adenylate cyclase-activating polypeptide (PACAP) in two inbred rat strains: stress hypo-responsive Lewis (LEW) and hyper-responsive Fisher 344 (F344) rats. We found site-specific and strain-dependent differences in the basal and stress-stimulated expression of 11HSD1, CRH, UCN2, UCN3 and PACAP. In LEW rats, stress upregulated 11HSD1 in the prefrontal cortex and lateral amygdala, whereas in F344 rats 11HSD1 was upregulated in the central amygdala and hippocampal CA2 and ventral but not dorsal CA1 region; no effect was observed in the paraventricular nucleus, pituitary gland and adrenal cortex of both strains. The expression of glucocorticoid receptors did not parallel the upregulation of 11HSD1. Stress also stimulated the expression of paraventricular OXT, CRH, UCN3 and PACAP in both strains but amygdalar CRH only in LEW and UCN2/UCN3 in F344 rats, respectively. The upregulation of PACAP and CRH was paralleled only by increased expression of PACAP receptor PAC1 but not CRH receptor type 1. These observations provide evidence that inbred F344 and LEW rats exhibit not only the well-known phenotypic differences in the activity of the HPA axis but also strain- and stress-dependent differences in the expression of genes encoding 11HSD1 and neuropeptides associated with the HPA axis activity. Moreover, the differences in 11HSD1 expression suggest different local concentration of corticosterone and access to GR in canonical and noncanonical structures of the HPA axis.

11ß-Hydroxysteroid dehydrogenase 1: Regeneration of active glucocorticoids is only part of the story.

11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) is an endoplasmic reticulum membrane enzyme with its catalytic site facing the luminal space. It functions primarily as a reductase, driven by the supply of its cosubstrate NADPH by hexose-6-phosphate dehydrogenase (H6PDH). Extensive research has been performed on the role of 11ß-HSD1 in the regeneration of active glucocorticoids and its role in inflammation and metabolic disease. Besides its important role in the fine-tuning of glucocorticoid action, 11ß-HSD1 is a multi-functional carbonyl reductase converting several 11- and 7-oxosterols into the respective 7-hydroxylated forms. Moreover, 11ß-HSD1 has a role in phase I biotransformation reactions and catalyzes the carbonyl reduction of several non-steroidal xenobiotics. Recent observations from experiments using selective inhibitors and studies with transgenic mice indicated a role for 11ß-HSD1 in oxysterol metabolism and in bile acid homeostasis, with evidence for glucocorticoid-independent effects on gene expression. This review focuses on the promiscuity of 11ß-HSD1 to accept structurally distinct substrates and discusses recent progress mainly on non-glucocorticoid substrates. This article is part of a Special Issue entitled 'Enzyme Promiscuity and Diversity'.

Regulation of 11ß-hydroxysteroid dehydrogenase type 1 and 7α-hydroxylase CYP7B1 during social stress.

11ß-hydroxysteroid dehydrogenase type 1 (11HSD1) is an enzyme that amplifies intracellular glucocorticoid concentration by the conversion of inert glucocorticoids to active forms and is involved in the interconversion of 7-oxo- and 7-hydroxy-steroids, which can interfere with the activation of glucocorticoids. The presence of 11HSD1 in the structures of the hypothalamic-pituitary-adrenal (HPA) axis suggests that this enzyme might play a role in the regulation of HPA output. Here we show that the exposure of Fisher 344 rats to mild social stress based on the resident-intruder paradigm increased the expression of 11HSD1 and CYP7B1, an enzyme that catalyzes 7-hydroxylation of steroids. We found that social behavioral profile of intruders was significantly decreased whereas their plasma levels of corticosterone were increased more than in residents. The stress did not modulate 11HSD1 in the HPA axis (paraventricular nucleus, pituitary, adrenal cortex) but selectively upregulated 11HSD1 in some regions of the hippocampus, amygdala and prelimbic cortex. In contrast, CYP7B1 was upregulated not only in the hippocampus and amygdala but also in paraventricular nucleus and pituitary. Furthermore, the stress downregulated 11HSD1 in the thymus and upregulated it in the spleen and mesenteric lymphatic nodes whereas CYP7B1 was upregulated in all of these lymphoid organs. The response of 11HSD1 to stress was more obvious in intruders than in residents and the response of CYP7B1 to stress predominated in residents. We conclude that social stress induces changes in enzymes of local metabolism of glucocorticoids in lymphoid organs and in brain structures associated with the regulation of the HPA axis. In addition, the presented data clearly suggest a role of 11HSD1 in modulation of glucocorticoid feedback of the HPA axis during stress.

Local metabolism of glucocorticoids in Prague hereditary hypertriglyceridemic rats--effect of hypertriglyceridemia and gender.

11ß-Hydroxysteroid dehydrogenase type 1 (11HSD1) is a microsomal NADPH-dependent oxidoreductase which elevates intracellular concentrations of active glucocorticoids. Data obtained from mouse strains with genetically manipulated 11HSD1 showed that local metabolism of glucocorticoids plays an important role in the development of metabolic syndrome. Tissue specific dysregulation of 11HSD1 was also found in other models of metabolic syndrome as well as in a number of clinical studies. Here, we studied local glucocorticoid action in the liver, subcutaneous adipose tissue (SAT) and skeletal muscles of male and female Prague hereditary hypertriglyceridemic rats (HHTg) and their normotriglyceridemic counterpart, the Wistar rats. 11HSD1 bioactivity was measured as a conversion of [(3)H]11-dehydrocorticosterone to [(3)H]corticosterone or vice versa. Additionally to express level of active 11HSD1 protein, enzyme activity was measured in tissue homogenates. mRNA abundance of 11HSD1, hexoso-6-phosphate dehydrogenase (H6PDH) and the glucocorticoid receptor (GR) was measured by real-time PCR. In comparison with normotriglyceridemic animals, female HHTg rats showed enhanced regeneration of glucocorticoids in the liver and the absence of any changes in SAT and skeletal muscle. In contrast to females, the glucocorticoid regeneration in males of HHTg rats was unchanged in liver, but stimulated in SAT and downregulated in muscle. Furthermore, SAT and skeletal muscle exhibited not only 11-reductase but also 11-oxidase catalyzed by 11HSD1. In females of both strains, 11-oxidase activity largely exceeded 11-reductase activity. No dramatic changes were found in the mRNA expression of H6PDH and GR. Our data provide evidence that the relationship between hypertriglyceridemia and glucocorticoid action is complex and gender specific.

Local metabolism of glucocorticoids and its role in rat adjuvant arthritis.

11beta-Hydroxysteroid dehydrogenase 1 (11HSD1) regulates local glucocorticoid activity and plays an important role in various diseases. Here, we studied whether arthritis modulates 11HSD1, what is the role of pro-inflammatory cytokines in this process and whether altered local metabolism of glucocorticoids may contribute to the feedback regulation of inflammation. Adjuvant arthritis increased synovial 11HSD1 mRNA and 11-reductase activity but treatments with tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) antagonists etanercept and anakinra reduced 11HSD1 upregulation. Treatment with carbenoxolone, an 11HSD inhibitor, increased expression of TNF-alpha, cyclooxygenase 2, and osteopontin mRNA without any changes in the plasma levels of corticosterone. Similar changes were observed when arthritic rats were treated with RU486, an antagonist of GR. This study suggests that arthritis upregulates synovial 11HSD1, this upregulation is controlled by TNF-alpha and IL-1beta and that the increased supply of local corticosterone might contribute to feedback regulation of inflammation.

Chronic intermittent hypoxia induces 11beta-hydroxysteroid dehydrogenase in rat heart.

Corticosteroids are known to not only regulate electrolyte homeostasis but also play a role in the cardiovascular system, including myocardial remodeling. Because transgenic mice that overexpress 11beta-hydroxysteroid dehydrogenase (11HSD) type 2 in cardiomyocytes have been shown to spontaneously develop cardiac hypertrophy and fibrosis, we investigated whether changes in the cardiac metabolism of glucocorticoids accompany remodeling of the heart under physiological conditions. In the present study, glucocorticoid metabolism and 11HSD2 were explored in the hearts of rats exposed to chronic intermittent hypobaric hypoxia (CIH), which induces hypertrophy and fibrosis of the right and less of the left ventricle. We first demonstrated that adaptation to CIH led to a significant increase in 11HSD2 transcript levels and activity in the myocardium. In contrast, neither 11HSD1 activity and mRNA level nor the abundance of mineralocorticoid and glucocorticoid receptor mRNA were up-regulated. The adaptation to CIH also led to an increase of 11HSD2 mRNA in isolated cardiomyocytes, whereas 11HSD1, glucocorticoid receptor, and mineralocorticoid receptor mRNA levels were not changed in comparison with the cardiomyocytes of control normoxic rats. The changes in cardiac metabolism of glucocorticoids were accompanied by inflammatory responses. The expression levels of the proinflammatory markers cyclooxygenase-2 and osteopontin were significantly increased in both the myocardium and the cardiomyocytes isolated from rats exposed to CIH. These findings suggest that myocardial remodeling induced by CIH is associated with the up-regulation of cardiac 11HSD2. Consequently, local metabolism of glucocorticoids could indeed play a role in cardiac hypertrophy and fibrosis.

Occurrence of lipids in the liver of the hypertriglyceridemic rats.

AIMS: The purpose of this study was to demonstrate the accumulation and distribution of lipids in the liver of the adult Prague hereditary hypertriglyceridemic (HHTg) rats. They reveal an increased expression of 11beta-hydroxysteroid dehydrogenase 1 (11HSD1), which locally increases concentration of corticosterone in the liver. We studied the effect of the 11HSD1 inhibition on the lipid content. METHODS: Samples of liver of three groups of adult female rats--HHTg, HHTg treated for 14 days with 50 mg/kg/day carbenoxolone (HHTg+CBX) and control Wistar rats, were examined histochemically. Cryosections of the samples were stained with Oil red O or Sudan black B to demonstrate different kinds of lipids. Extent and intensity of staining was evaluated semiquantitatively. RESULTS: The orientational analysis showed a higher extent and intensity of the staining of the liver of HHTg and HHTg+CBX rats (equal in both hypertriglyceridemic groups) than that of the control Wistar rats. Oil red O stained unsaturated fatty acids and neutral fats, mainly triglycerides. The difference was on average 30 per cent. Staining of phospholipids with Sudan black B showed similarly the higher positivity in the hypertriglyceridemic groups than in controls. CONCLUSIONS: Staining for triglycerides and phospholipids demonstrated a higher amount of lipids in the liver of HHTg and HHTg+CBX female rats than in controls. The inhibition of 11HSD1 activity had no effect on the lipid content in the liver of the HHTg rats.

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.

Chicken 11beta-hydroxysteroid dehydrogenase type 2: partial cloning and tissue distribution.

NAD(+)-dependent 11beta-hydroxysteroid dehydrogenase (11HSD2) converts glucocorticoids to 11-oxo derivatives and thus decreases their local concentration and prevents them from activating corticosteroid receptors. In this paper we report the partial cloning, characterization and tissue distribution of chicken 11HSD2. A cDNA of 991bp was cloned from kidney mRNA by reverse transcription and polymerase chain reaction. At the amino acid level, the sequence of PCR product had 56-59% homology with mammalian and 46-48% with fish 11HSD2. The consensus sequences of the short-chain dehydrogenase/reductase superfamily such as the catalytic activity motif Tyr-X-X-X-Lys and cosubstrate-binding motif Gly-X-X-X-Gly-X-Gly, were found in the cloned cDNA. Analysis of the tissue expression of chicken 11HSD2 mRNA and NAD(+)-dependent 11beta-oxidase activity showed a similar tissue distribution pattern in the majority of tissues. High levels of expression and activity were found in kidney, small intestine, colon and oviduct; low in ovary and almost zero in brain, liver and testis.