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  • br Materials and methods br

    2022-01-05


    Materials and methods
    Results
    Discussion In this study, we demonstrated, for the first time, that manipulation of AKR1D1 expression and activity is able to regulate glucocorticoid availability in liver and non-liver cell lines. AKR1D1 over-expression leads to increased glucocorticoid clearance and decreased GR activation, whilst AKR1D1 knockdown is able to decrease glucocorticoid clearance in human hepatocytes. In addition, we showed that finasteride and dutasteride, two 5αR inhibitors, failed to prevent AKR1D1-induced glucocorticoid clearance, in either cell-free or in vitro cellular systems. The crystal structure and enzymology of human AKR1D1 has been extensively characterized [14,26]. AKR1D1 has broad substrate specificity [15]. Disease-causing missense mutations lead to decreased functional activity and when they occur in evolutionary conserved amino acids, enzyme stability is impaired [15,27]. Patients with mutations in AKR1D1 present with a rare and potentially severe form of neonatal cholestasis and can be treated effectively with bile 5ar inhibitors replacement [28]. However, survival into adulthood in the absence of treatment is reported. The role of AKR1D1 in steroid hormone metabolism in vitro and in vivo has not been explored in detail. The urinary steroid profile in a single patient with a missense mutation in AKR1D1 (662C > T resulting in a Pro198Leu substitution) provided the first in vivo evidence of the ability of AKR1D1 to alter steroid hormone availability with the potential to alter glucocorticoid availability and action. In this patient, there was a marked reduction of all 5β-reduced steroid hormone metabolites, and in particular, an absence of 5β-tetrahydrocortisol [29]. There was a partial compensatory increase in 5α-reduced metabolites. Our findings in human hepatoma cells suggest that AKR1D1 expression may act as a protective mechanism against glucocorticoid excess by not only reducing cortisol within the liver, but also limiting the generation of cortisol from cortisone [through enhanced clearance of substrate (cortisone) for 11β-HSD1]. In this regard, there are important parallels with the isozymes of 11β-HSD and 5αR. Genetic and pharmacological manipulation of these systems alters steroid hormone availability in a tissue-specific manner that is independent of circulating steroid hormone levels. In addition, changes in activity and expression of 11β-HSDs and 5αRs have been linked to the development of metabolic diseases including NAFLD, T2DM and insulin resistance [12,30,31]. Furthermore, clinical studies using pharmacological inhibitors have shown improvements in clinical phenotype with selective 11β-HSD1 inhibitors [[32], [33], [34]] and a detrimental effect of 5αR inhibitors [35,36] putatively through decreasing and increasing tissue-specific cortisol availability, respectively. In this study, we have focused exclusively on the role of AKR1D1 to metabolize endogenous glucocorticoids. Whilst this is clearly important in understanding its physiological role, there is a broader context in that 2–3% of the population of the UK and USA are prescribed therapeutic glucocorticoids, including dexamethasone, prednisone and prednisolone [37,38]. It is well established that synthetic glucocorticoids are poorly metabolized within human tissues, and demonstrate poor cortisol binding globulin affinity and slower plasma removal rates, thus having more prolonged action when compared to endogenous glucocorticoids [[39], [40], [41]]. However, several studies have shown that synthetic glucocorticoids can also be inactivated through a variety of metabolic pathways, including oxidation or reduction by CYP3A4, isoforms of 11β-HSD and 5αR [[42], [43], [44], [45]]. In this regard, the ability of AKR1D1 is entirely unexplored, but is likely to be of equal importance. In addition, little is currently known about the ability of glucocorticoids in the regulation of expression and activity of AKR1D1.