Glucocorticoids regulate AKR1D1 activity in human liver in vitro and in vivo

Abstract

Steroid 5β-reductase (AKR1D1) is highly expressed in human liver where it inactivates endogenous glucocorticoids and catalyses an important step in bile acid synthesis. Endogenous and synthetic glucocorticoids are potent regulators of metabolic phenotype and play a crucial role in hepatic glucose metabolism. However, the potential of synthetic glucocorticoids to be metabolised by AKR1D1 as well as to regulate its expression and activity has not been investigated. The impact of glucocorticoids on AKR1D1 activity was assessed in human liver HepG2 and Huh7 cells; AKR1D1 expression was assessed by qPCR and Western blotting. Genetic manipulation of AKR1D1 expression was conducted in HepG2 and Huh7 cells and metabolic assessments were made using qPCR. Urinary steroid metabolite profiling in healthy volunteers was performed pre- and post-dexamethasone treatment, using gas chromatography-mass spectrometry. AKR1D1 metabolised endogenous cortisol, but cleared prednisolone and dexamethasone less efficiently. In vitro and in vivo, dexamethasone decreased AKR1D1 expression and activity, further limiting glucocorticoid clearance and augmenting action. Dexamethasone enhanced gluconeogenic and glycogen synthesis gene expression in liver cell models and these changes were mirrored by genetic knockdown of AKR1D1 expression. The effects of AKR1D1 knockdown were mediated through multiple nuclear hormone receptors, including the glucocorticoid, pregnane X and farnesoid X receptors. Glucocorticoids down-regulate AKR1D1 expression and activity and thereby reduce glucocorticoid clearance. In addition, AKR1D1 down-regulation alters the activation of multiple nuclear hormone receptors to drive changes in gluconeogenic and glycogen synthesis gene expression profiles, which may exacerbate the adverse impact of exogenous glucocorticoids.

Differential activity and expression of human 5β-reductase (AKR1D1) splice variants

“…In a clinical study, urinary steroid metabolome analysis of samples from healthy male volunteers, following prednisolone administration, identified 20 different prednisolone metabolites, including 11-hydroxylated, 20-reduced and 5α/β-reduced products ( Matabosch et al 2015 ). Supporting these findings, we have recently demonstrated the ability of AKR1D1-002 to clear prednisolone and dexamethasone, with concomitant decreases in hepatic GR activation ( Nikolaou et al 2019 a , 2020 ). We now show that, in addition to AKR1D1-002, AKR1D1-001 and AKR1D1-006 metabolise dexamethasone (albeit poorly) in vitro , but this poor clearance did not result in any significant differences in GR activation.…”

“…The 5β-reduced metabolites are then converted, in a non-rate limiting step, to their inactive tetrahydro-metabolites (5β-tetrahydrocortisol and 5β-tetrahydrocortisone) by the hepatic 3α-hydroxysteroid dehydrogenases, AKR1C1-C4, with downstream effects on steroid receptor activation and target gene transcription ( Chen et al 2011 , Nikolaou et al 2019 a ). Crucially, AKR1D1-002 is also implicated in drug metabolism, as it metabolises the synthetic glucocorticoids prednisolone and dexamethasone, with downstream decreases in glucocorticoid receptor activation ( Nikolaou et al 2020 ).…”

Differential activity and expression of human 5β-reductase (AKR1D1) splice variants

“…In a clinical study, urinary steroid metabolome analysis of samples from healthy male volunteers, following prednisolone administration, identified 20 different prednisolone metabolites, including 11-hydroxylated, 20-reduced and 5α/β-reduced products (Matabosch et al 2015). Supporting these findings, we have recently demonstrated the ability of AKR1D1-002 to clear prednisolone and dexamethasone, with concomitant decreases in hepatic GR activation (Nikolaou et al 2019a, 2020). We now show that, in addition to AKR1D1-002, AKR1D1-001 and AKR1D1-006 metabolise dexamethasone (albeit poorly) in vitro .…”

“…The 5β-reduced metabolites are then converted, in a non-rate limiting step, to their inactive tetrahydro-metabolites (5β-tetrahydrocortisol and 5β-tetrahydrocortisone) by the hepatic 3α-hydroxysteroid dehydrogenases, AKR1C1-C4, with downstream effects on steroid receptor activation and target gene transcription (Chen et al 2011; Nikolaou et al 2019a). Crucially, AKR1D1-002 is also implicated in drug metabolism, as it metabolises the synthetic glucocorticoids prednisolone and dexamethasone, leading to the formation of inactive 5β-reduced products (Nikolaou et al 2020).…”

Akr1d1-/- mice have a sexually dimorphic metabolic phenotype with reduced fat mass, increased insulin sensitivity and hypertriglyceridemia in males

“…This phenotype is in contrast to Cyp7a1-/- mice which, although mirroring the insulin sensitivity of Akr1d1-/- males, have normal liver triacylglycerol and serum lipids 41 . In human hepatoma cells, the effects of AKR1D1 knockdown on the expression of lipid metabolism genes were partially recovered by LXRα, LXRβ and PXR antagonism as well as FXR agonism 18–20 suggesting accumulation of oxysterols and bioactive intermediates of bile acid synthesis. However, in Akr1d1-/- males we did not observe any differences in 27-hydroxycholesterol or the AKR1D1 substrates 7α-12α-dihydroxy-4-chol-3-one and 7α-hydroxy-4-chol-3-one, and Ingenuity Pathway Analysis did not identify hepatic gene expression signatures associated with oxysterols, bile acids or bile acid intermediates.…”