Impaired oxidoreduction by 11β-hydroxysteroid dehydrogenase 1 results in the accumulation of 7-oxolithocholic acid
2013; Elsevier BV; Volume: 54; Issue: 10 Linguagem: Inglês
10.1194/jlr.m042499
ISSN1539-7262
AutoresCarlos A. Penno, Stuart Morgan, Anna Vuorinen, Daniela Schuster, Gareth G. Lavery, Alex Odermatt,
Tópico(s)Pharmacological Effects of Natural Compounds
Resumo11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) mediates glucocorticoid activation and is currently considered as therapeutic target to treat metabolic diseases; however, biomarkers to assess its activity in vivo are still lacking. Recent in vitro experiments suggested that human 11β-HSD1 metabolizes the secondary bile acid 7-oxolithocholic acid (7-oxoLCA) to chenodeoxycholic acid (CDCA) and minor amounts of ursodeoxycholic acid (UDCA). Here, we provide evidence from in vitro and in vivo studies for a major role of 11β-HSD1 in the oxidoreduction of 7-oxoLCA and compare its level and metabolism in several species. Hepatic microsomes from liver-specific 11β-HSD1-deficient mice were devoid of 7-oxoLCA oxidoreductase activity. Importantly, circulating and intrahepatic levels of 7-oxoLCA and its taurine conjugate were significantly elevated in mouse models of 11β-HSD1 deficiency. Moreover, comparative enzymology of 11β-HSD1-dependent oxidoreduction of 7-oxoLCA revealed that the guinea-pig enzyme is devoid of 7-oxoLCA oxidoreductase activity. Unlike in other species, 7-oxoLCA and its glycine conjugate are major bile acids in guinea-pigs. In conclusion, the oxidoreduction of 7-oxoLCA and its conjugated metabolites are catalyzed by 11β-HSD1, and the lack of this activity leads to the accumulation of these bile acids in guinea-pigs and 11β-HSD1-deficient mice. Thus, 7-oxoLCA and its conjugates may serve as biomarkers of impaired 11β-HSD1 activity. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) mediates glucocorticoid activation and is currently considered as therapeutic target to treat metabolic diseases; however, biomarkers to assess its activity in vivo are still lacking. Recent in vitro experiments suggested that human 11β-HSD1 metabolizes the secondary bile acid 7-oxolithocholic acid (7-oxoLCA) to chenodeoxycholic acid (CDCA) and minor amounts of ursodeoxycholic acid (UDCA). Here, we provide evidence from in vitro and in vivo studies for a major role of 11β-HSD1 in the oxidoreduction of 7-oxoLCA and compare its level and metabolism in several species. Hepatic microsomes from liver-specific 11β-HSD1-deficient mice were devoid of 7-oxoLCA oxidoreductase activity. Importantly, circulating and intrahepatic levels of 7-oxoLCA and its taurine conjugate were significantly elevated in mouse models of 11β-HSD1 deficiency. Moreover, comparative enzymology of 11β-HSD1-dependent oxidoreduction of 7-oxoLCA revealed that the guinea-pig enzyme is devoid of 7-oxoLCA oxidoreductase activity. Unlike in other species, 7-oxoLCA and its glycine conjugate are major bile acids in guinea-pigs. In conclusion, the oxidoreduction of 7-oxoLCA and its conjugated metabolites are catalyzed by 11β-HSD1, and the lack of this activity leads to the accumulation of these bile acids in guinea-pigs and 11β-HSD1-deficient mice. Thus, 7-oxoLCA and its conjugates may serve as biomarkers of impaired 11β-HSD1 activity. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a NADPH-dependent enzyme catalyzing the regeneration of cortisol (in humans, higher mammals) and corticosterone (in rodents) from inactive cortisone and 11-dehydrocorticosterone, respectively, thereby controlling the tissue- and cell-specific exposure to active glucocorticoids (1Odermatt A. Atanasov A.G. Balazs Z. Schweizer R.A.S. Nashev L.G. Schuster D. Langer T. Why is 11β-hydroxysteroid dehydrogenase type 1 facing the endoplasmic reticulum lumen? Physiological relevance of the membrane topology of 11β-HSD1.Mol. Cell. Endocrinol. 2006; 248: 15-23Crossref PubMed Scopus (64) Google Scholar). 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Furthermore, an ideal biomarker should be independent of mechanisms of negative feedback regulation, such as the control of circulating glucocorticoid levels by the hypothalamus-pituitary-adrenal (HPA) axis and stress-induced fluctuations (18Harno E. White A. Will treating diabetes with 11β-HSD1 inhibitors affect the HPA axis?.Trends Endocrinol. Metab. 2010; 21: 619-627Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 19Odermatt A. Nashev L.G. The glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 has broad substrate specificity: Physiological and toxicological considerations.J. Steroid Biochem. Mol. Biol. 2010; 119: 1-13Crossref PubMed Scopus (38) Google Scholar). 11β-HSD1 has broad substrate specificity and metabolizes, besides 11-ketoglucocorticoids, several oxidized endogenous steroids and sterols (19Odermatt A. Nashev L.G. 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In the present study, we aimed to i) obtain further in vitro and in vivo evidence for a role of 11β-HSD1 in the oxidoreduction of 7-oxoLCA, ii) assess species-specific differences in the 11β-HSD1-dependent oxidoreduction of 7-oxoLCA, and iii) test our hypothesis that 7-oxoLCA might accumulate in transgenic 11β-HSD1-deficient mice and provide initial evidence that this bile acid might serve as a biomarker for 11β-HSD1 deficiency. CDCA, UDCA, [2,2,4,4-2H4]CDCA (CDCA-d4) (>98% isotopic purity), and [9,11,12,12-2H4]cortisol (cortisol-d4) (>98% isotopic purity) were purchased from Sigma-Aldrich (St. Louis, MO). 7-oxolithocholic acid (7-oxoLCA) was purchased from Steraloids (Newport, RI). 7-oxolithocholyltaurine (7-oxoLC-Tau) and 7-oxolithocholylglycine (7-oxoLC-Gly) were a kind gift from Dr. Alan F. Hofmann (University of California, San Diego, CA). Cell culture media were purchased from Invitrogen (Carlsbad, CA) and Sigma (Buchs, Switzerland). [1,2-3H]cortisone was obtained from American Radiolabeled Chemicals (St. Louis, MO), [1,2,6,7-3H]cortisol from Amersham Pharmacia (Piscataway, NJ), and tricyclo[3.3.1.13,7] dec-1-yl-6,7,8,9-tetrahydro-5H-1,2,4-triazolo[4,3-a]azepine (T0504) from Enamine (Kiev, Ukraine). The construction of expression plasmids has been described earlier (29Arampatzis S. Kadereit B. Schuster D. Balazs Z. Schweizer R.A.S. Frey F.J. Langer T. Odermatt A. Comparative enzymology of 11β-hydroxysteroid dehydrogenase type 1 from six species.J. Mol. Endocrinol. 2005; 35: 89-101Crossref PubMed Scopus (67) Google Scholar, 30Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. The N-terminal anchor sequences of 11β-hydroxysteroid dehydrogenases determine their orientation in the endoplasmic reticulum membrane.J. Biol. Chem. 1999; 274: 28762-28770Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Male human and guinea-pig liver microsomes were obtained from Celsis International Ltd. (Brussels, Belgium), and serum samples from 12–16 h fasted adult male mice (balb/c and C57bL/6), rats (Han Wistar and Sprague-Dawley), dog (Canis familiaris, beagle bred), guinea-pigs (Dunkin-Hartley), and hamsters (golden Syrian) were obtained from Harlan (Gannat, France). To assess species-dependent differences in the 11β-HSD1-dependent metabolism of 7-oxoLCA, we transiently transfected HEK-293 (human embryonic kidney-293) cells [cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 units/ml penicillin, 50 μg/ml streptomycin and 2 mM glutamine] with plasmids coding for human, rat, mouse, hamster, canine, and guinea-pig 11β-HSD1 as described previously (23Schweizer R.A.S. Zürcher M. Balazs Z. Dick B. Odermatt A. Rapid hepatic metabolism of 7-ketocholesterol by 11β-hydroxysteroid dehydrogenase type 1.J. Biol. Chem. 2004; 279: 18415-18424Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 29Arampatzis S. Kadereit B. Schuster D. Balazs Z. Schweizer R.A.S. Frey F.J. Langer T. Odermatt A. Comparative enzymology of 11β-hydroxysteroid dehydrogenase type 1 from six species.J. Mol. Endocrinol. 2005; 35: 89-101Crossref PubMed Scopus (67) Google Scholar, 30Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. The N-terminal anchor sequences of 11β-hydroxysteroid dehydrogenases determine their orientation in the endoplasmic reticulum membrane.J. Biol. Chem. 1999; 274: 28762-28770Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). At 48 h posttransfection (jetPRIME, Polyplus transfection, Illkirch, France) cells were detached and centrifuged, and pellets were stored at -80°C. Cell pellets were resuspended in TS2 buffer (100 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM MgCl2, 250 mM sucrose, and 20 mM Tris/HCl, pH 7.4), sonicated, and used immediately to measure enzyme activity. To determine apparent Km and Vmax values, lysates were incubated for 10 min at 37°C in a total volume of 20 μl containing 500 μM NADPH and 7-oxoLCA at concentrations between 62.5 nM and 4 μM. To determine the relative content of product formed, lysates at a high total protein concentration (to achieve almost complete substrate conversion) were incubated with 1 µM of 7-oxoLCA for 10 min. Reactions were terminated by adding 80 µl of acetonitrile containing 100 nM of CDCA-d4. Concomitantly, lysates were used in parallel experiments to assess the conversion of cortisone to cortisol. Reaction products were directly measured by mass spectrometry as described previously (26Odermatt A. Da Cunha T. Penno C.A. Chandsawangbhuwana C. Reichert C. Wolf A. Dong M. Baker M.E. Hepatic reduction of the secondary bile acid 7-oxolithocholic acid is mediated by 11beta-hydroxysteroid dehydrogenase 1.Biochem. J. 2011; 436: 621-629Crossref PubMed Scopus (37) Google Scholar, 31Penno C.A. Arsenijevic D. Da Cunha T. Kullak-Ublick G.A. Montani J-P. Odermatt A. Quantification of multiple bile acids in uninephrectomized rats using ultra-performance liquid chromatography-tandem mass spectrometry.Anal. Methods. 2013; 5: 1155-1164Crossref Scopus (13) Google Scholar). The rate of all reactions was kept below 25% of substrate conversion, with the exception of the comparison of product formation from 7-oxoLCA shown in Fig. 1A. The expression of recombinant C-terminally FLAG-tagged 11β-HSD1 in different transfection experiments was determined semiquantitatively by immunoblotting. Protein concentrations were determined by the bicinchoninic acid (BCA) method. An amount of 25 µg of total protein was resolved on 12% Bis-Tris gels (NuPage®, Invitrogen) using 1X MES as buffer (Invitrogen, NuPAGE® MES SDS Running Buffer) and then transferred to nitrocellulose membranes (iBlot®, Invitrogen). Thereafter, membranes were blocked with Odyssey® blocking buffer (LI-COR, Biosciences, Lincoln, NE) overnight at 4°C. Immunoreactions were carried out with primary anti-FLAG antibody M2 (Invitrogen) to detect the recombinant enzyme and with secondary goat anti-mouse Alexa Fluor® 790 antibody, respectively (Invitrogen). After immunoreaction of FLAG-tagged enzymes, the membranes were stripped and incubated with an antibody against β-actin (Sigma). All detection and quantification reactions were performed using a LI-COR Odyssey infrared imaging system (LI-COR, Biosciences, Lincoln, NE). The inhibitory impact of bile acids on the interconversion of glucocorticoids by 11β-HSD1 of six species was performed as described previously (26Odermatt A. Da Cunha T. Penno C.A. Chandsawangbhuwana C. Reichert C. Wolf A. Dong M. Baker M.E. Hepatic reduction of the secondary bile acid 7-oxolithocholic acid is mediated by 11beta-hydroxysteroid dehydrogenase 1.Biochem. J. 2011; 436: 621-629Crossref PubMed Scopus (37) Google Scholar). Briefly, lysates were incubated for 10 min at 37°C in a total volume of 22 μl containing 200 nM and 10 nCi of [1,2-3H]cortisone or [1,2,6,7-3H]cortisol and 500 μM cofactor NADPH or NADP+, respectively, and vehicle or various concentrations of bile acids. Following the interconversion of radiolabeled glucocorticoids and termination of reactions by adding methanol containing 2 mM of unlabeled cortisone and cortisol, 15 μl were spotted on Polygram SIL G-25 UV254 silica plates (Macherey-Nagel, Oensingen, Switzerland). Plates were dried, and cortisone and cortisol were resolved using a solvent system of 9:1 (v/v) chloroform/methanol. The separated steroids were analyzed by scintillation counting. Enzyme kinetics was analyzed by nonlinear regression using four-parameter logistic curve fitting. For statistical comparisons, the ratio t-test in GraphPad Prism 5 software was used. Results (mean ± SD) were obtained from at least three independent experiments. For calculation of Vmax, the expression level of the FLAG-tagged enzyme was normalized to the expression signal of the internal control β-actin. Microsomes from livers of wild-type and liver-specific 11β-HSD1-knockout mice were prepared as described earlier (32Senesi S. Legeza B. Balázs Z. Csala M. Marcolongo P. Kereszturi é. Szelényi P. Egger C. Fulceri R. Mandl J. et al.Contribution of fructose-6-phosphate to glucocorticoid activation in the endoplasmic reticulum: possible implication in the metabolic syndrome.Endocrinology. 2010; 151: 4830-4839Crossref PubMed Scopus (30) Google Scholar). The quality of microsomal preparations was validated by using a kit to measure cytochrome c reductase activity (Sigma, Saint Louis, MO). 11β-HSD1 reductase activity was determined by incubation of microsomes (0.05 mg/ml) for 60 min at 37°C in a total volume of 25 μl containing TS2 buffer, 500 μM NADPH or 1 mM glucose-6-phosphate (G6P), 1 μM 7-oxoLCA or 1 µM cortisone, and vehicle or 5 μM of the 11β-HSD1 inhibitor T0504 as indicated. Substrates and inhibitors were diluted from 10 mM stock solutions in DMSO or methanol. The final solvent concentration in all reactions was kept below 0.2%. Reactions were started by adding microsomes into freshly prepared reaction mixture and stopped by adding 500 µl of acetonitrile containing CDCA-d4 and cortisol-d4 at a concentration of 100 nM as internal standards for LC-MS/MS analysis. Thereafter, the organic phase was evaporated to dryness, and samples were reconstituted in 50% methanol/water solution, followed by injection into the LC-MS/MS instrument. Animal studies were conducted under Home Office license and following approval of the Joint Ethics and Research Governance Committee of the University of Birmingham (Birmingham, United Kingdom) in accordance with the UK Animals (Scientific Procedures) Act, 1986. Mice were kept in a climate-controlled facility, housed under standard conditions on a 12 h light/dark cycle, and fed ad libitum with standard chow and free access to drinking water. To assess the impact of 11β-HSD1 on the circulating and intrahepatic levels of 7-oxoLCA and its conjugated metabolites, 15-week-old wild-type mice (n = 16), 11β-HSD1 global KO (n = 8), and 11β-HSD1 liver-specific KO (n = 16), previously described in Ref. 33Lavery G.G. Zielinska A.E. Gathercole L.L. Hughes B. Semjonous N. Guest P. Saqib K. Sherlock M. Reynolds G. Morgan S.A. et al.Lack of significant metabolic abnormalities in mice with liver-specific disruption of 11β-hydroxysteroid dehydrogenase type 1.Endocrinology. 2012; 153: 3236-3248Crossref PubMed Scopus (57) Google Scholar, were fasted overnight, and blood samples were collected by intracardiac puncture. Plasma was prepared immediately by centrifugation, and samples were stored at -80°C until further processing. Extraction and quantification of bile acids in plasma samples was performed essentially as described previously (31Penno C.A. Arsenijevic D. Da Cunha T. Kullak-Ublick G.A. Montani J-P. Odermatt A. Quantification of multiple bile acids in uninephrectomized rats using ultra-performance liquid chromatography-tandem mass spectrometry.Anal. Methods. 2013; 5: 1155-1164Crossref Scopus (13) Google Scholar). The extraction of bile acids from liver tissue was performed by homogenizing 100 mg of tissue in 200 µl 50% methanol. Samples were spiked with 300 µl of deuterium labeled bile acids as internal standards (CDCA-d4) at a final concentration of 1 µM in acetonitrile, followed by protein precipitation by adding 1.5 ml of alkaline (5% NH4OH) ice-cold acetonitrile. Thereafter, samples were mixed continuously for 1 h and centrifuged at 11,000 g for 10 min. The supernatant was transferred to a new tube, the solvent was evaporated, and the residue was reconstituted in 100 µl of 50% methanol, followed by an additional centrifugation step to remove insoluble particles. The method was qualified on the basis of extraction efficiency, intraday accuracy, and precision for 7-oxoLCA, 7-oxoLC-Tau, and 7-oxoLC-Gly (data not shown). The method presented acceptable extraction efficiency, accuracy, and precision for the bile acids studied. Data are presented as mean ± SD. Statistical significance was assessed by Student t-test. P≤ 0.05 was considered significant. The ligands were drawn using ChemBioDraw Ultra 12.0 and the 2D structures converted into 3D structures using ChemBio3D Ultra 12.0 (1986–2010 CambridgeSoft). The docking studies were performed using GOLD (34Verdonk M.L. Cole J.C. Hartshorn M.J. Murray C.W. Taylor R.D. Improved protein-ligand docking using GOLD.Proteins. 2003; 52: 609-623Crossref PubMed Scopus (2257) Google Scholar, 35Jones G. Willett P. Glen R.C. Leach A.R. Taylor R. Development and validation of a genetic algorithm for flexible docking.J. Mol. Biol. 1997; 267: 727-748Crossref PubMed Scopus (5365) Google Scholar), which uses a genetic algorithm to predict binding modes for small molecules in a protein binding site. The crystal structures for human and guinea pig 11β-HSD1 were downloaded from the Protein Data Bank (PDB, www.pdb.org) (36Berman H.M. Westbrook J. Feng Z. Gililand G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. The Protein Data Bank.Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27325) Google Scholar). PDB-entry 2BEL, chain A (37WuX.KavanaghK.SvenssonS.EllebyB.HultM.Von DelftF.MarsdenB.JornvallH.AbrahmsenL.OppermannU.. The high resolution structures of human, murine and guinea pig 11β-hydroxysteroid dehydrogenase type 1 reveal critical differences in active site architecture. [no journal] Epub ahead of print. 2004;doi:10.2210/pdb2bel/pdb.Google Scholar), was chosen for human protein, and 3LZ6, chain A (38Cheng H. Hoffman J. Le P. Nair S.K. Cripps S. Matthews J. Smith C. Yang M. Kupchinsky S. Dress K. et al.The development and SAR of pyrrolidine carboxamide 11β-HSD1 inhibitors.Bioorg. Med. Chem. Lett. 2010; 20: 2897-2902Crossref PubMed Scopus (36) Google Scholar), for guinea-pig protein. For human 11β-HSD1, the binding site was defined as an 8 Å sphere, centered with hydroxyl-oxygen of Ser170 (x: 3.84, y: 22.49, z: 13.34). For the guinea-pig protein, the binding site was defined as an 8 Å sphere, centered by hydroxyl-oxygen of Tyr158 (x: 13.21, y: 22.34, z: 45.45). For both enzymes, GoldScore was used as a scoring function, and the program was set to define the atom types for proteins and ligands automatically. The proteins were kept rigid and ligands flexible during the docking run. To give the steroidal ligands more flexibility, the program was set to flip ring corners while searching possible binding orientations for the ligands. For each ligand, a maximum of ten binding orientations were generated, but in case the three best-ranked solutions were within RMSD of 1 Å of each other, the program was allowed to terminate the run earlier. Using these settings, the program successfully reproduced the binding orientations of the co-crystallized ligands carbenoxolone (2bel) and N-adamantan-2-yl-1-ethyl-D-prolinamide (3lz6), thus validating the docking settings. We previously showed that human 11β-HSD1 preferentially converts 7-oxoLCA to CDCA (26Odermatt A. Da Cunha T. Penno C.A. Chandsawangbhuwana C. Reichert C. Wolf A. Dong M. Baker M.E. Hepatic reduction of the secondary bile acid 7-oxolithocholic acid is mediated by 11beta-hydroxysteroid dehydrogenase 1.Biochem. J. 2011; 436: 621-629Crossref PubMed Scopus (37) Google Scholar). Evidence from studies using rat liver microsomes indicated the formation of both CDCA and UDCA from 7-oxoLCA. In the present study, we investigated whether these observations are due to species-specific differences in the stereoselective product formation by 11β-HSD1 or whether additional enzymes might be involved in the carbonyl reduction of 7-oxoLCA in rats and mice. Recombinant 11β-HSD1 from six species were expressed in HEK-293 cells, and the carbonyl reduction of 7-oxoLCA was determined by incubation of lysates at high total protein concentration with 1 µM of 7-oxoLCA for 10 min to convert most of the substrate and to assess the relative composition of products formed. Human 1
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