Penno, Carlos Alberto.
The role of 11β-hydroxysteroid dehydrogenase type 1 in bile acid homeostasis.
PhD Thesis, University of Basel,
Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_10564
Bile acids (BAs) are important modulators of metabolic functions such as lipid, triglyceride and glucose homeostasis. Intrahepatic accumulation of BAs is known to cause liver injury in cholestatic conditions, where normal trans-hepatic BA flow is impaired due to pathological conditions or induced by toxic drugs. Therefore, it is important to understand the mechanisms of BA homeostasis regulation and to identify novel players and characterize their functions. The main goal of the present work was to investigate the impact of altered hepatic glucocorticoid activation by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) on BA homeostasis and to unravel the mechanisms of adaptations in a scenario of impaired 11β-HSD1 function. In order to achieve this goal, we developed and validated an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the quantification of a total of 24 BAs, including 11 unconjugated, 6 glycine-conjugated and 7 taurine-conjugated BAs, in biological matrices (serum/plasma and tissues) and cell culture supernatants. This method was validated and applied in a side project in which potential time-dependent changes of BAs in plasma from sham-operated and uninephrectomized male Sprague-Dawley rats were investigated. Several primary and secondary BAs were transiently elevated one week after uninephrectomy, followed by normalization thereafter. Using liver microsomal preparations and recombinant enzyme we then tested whether 11β-HSD1, in addition to its well-known role in the conversion of inactive to active glucocorticoids, is able to reduce 7-oxo BAs. We found that human 11β-HSD1 reduces the secondary BA 7-oxolithocholic acid (7-oxoLCA) mainly to chenodeoxycholic acid (CDCA) and to lesser amount to ursodeoxycholic acid (UDCA). 11β-HSD1 exclusively catalyzed the oxoreduction of 7-oxoLCA, in contrast to its role in the interconversion of glucocorticoids. The enzyme also metabolized 7-oxoLCA-glycine and -taurine conjugates. Furthermore, we compared 7-oxoLCA metabolism by human 11β-HSD1 with that of other species, including canine, guinea-pig, rat, mouse and hamster and we observed species-specific differences. While recombinant mouse and rat 11β-HSD1 converted 7-oxoLCA to equivalent amounts of CDCA and UDCA, the hamster and canine enzymes were stereoselective, producing mainly CDCA similar to the human enzyme. Surprisingly, guinea-pig 11β-HSD1 did not reduce 7-oxoLCA. The analysis of circulating BA profiles of several species confirmed earlier observations by other investigators, that 7-oxoLCA and its glycine-conjugated (G-7-oxoLCA) metabolite are abundant BAs in guinea-pigs compared with other species. These findings suggest that the lack of 11β-HSD1 oxoreductase activity on 7-oxoLCA in guinea-pigs is responsible for its elevated circulating levels. Next, we hypothesized that 7-oxoLCA might be a biomarker of impaired 11β-HSD1 activity. Analysis of BAs in serum from liver-specific 11β-HSD1 deficient mice revealed 18-, 47- and 7-fold elevation of 7-oxoLCA, its taurine and glycine conjugates compared with wild-type mice, respectively. In addition, 7-oxoLCA and its taurine conjugate were 2- and 6-fold elevated in liver from liver-specific 11β-HSD1 deficient mice. Moreover, BA profiles in serum and liver of liver-specific 11β-HSD1 deficient mice indicated a disturbed BA homeostasis. Circulating and intrahepatic levels of several unconjugated BAs species were up to 16-fold significantly elevated in liver-specific 11β-HSD1 deficient compared with wild-type mice. To pinpoint the molecular mechanism of altered BAs profiles, gene expression analysis was performed. The results suggest FXR-dependent decrease of BA synthesis, a compensatory effect to counteract the intrahepatic accumulation of BAs. In addition, the enzymes responsible for BA conjugation with coenzyme A, an intermediate step in BAs amidation, named VLCS and VLCSH2 displayed significantly lower expression levels in liver from liver-specific 11β-HSD1 deficient compared with wild-type mice. The decreased BA conjugating machinery in the liver of liver-specific 11β-HSD1 deficient mice may account for the elevated intrahepatic levels of unconjugated BAs observed. Moreover, the expression of OATP4, a basolateral BA transporter responsible for the uptake of unconjugated BAs from the circulation into hepatocytes presented reduced expression levels and may account for the significant elevation of several circulating unconjugated BAs found in liver-specific 11β-HSD1 deficient mice.
In conclusion, we demonstrated an important role of 11β-HSD1 in the oxoreduction on 7-oxoLCA and provided evidences that 7-oxoLCA its taurine conjugate are functional biomarkers of impair 11β-HSD1 activity. Circulating concentrations of these 7-oxo BAs may find application in the assessment of the therapeutic efficacy of 11β-HSD1 inhibitors. Moreover, we described for the first time the impact of intrahepatic glucocorticoid regeneration deficiency on BA homeostasis in mice. Our findings indicate that 11β-HSD1 is an important modulator of BAs homeostasis, and potential disturbances of BA homeostasis must be taken into account when assessing the safety of 11β-HSD1 inhibitors, with particular attention to cholestatic patients and patients receiving combined therapeutic regimens with drugs known to induce liver injury.
|Committee Members:||Arand, Michael|
|Faculties and Departments:||05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Pharmazie > Molecular and Systems Toxicology (Odermatt)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||120 Bl.|
|Last Modified:||30 Jun 2016 10:54|
|Deposited On:||08 Nov 2013 16:18|
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