Human Δ4-3-oxosteroid 5β-reductase (AKR1D1) deficiency and steroid metabolism

doi:10.1016/j.steroids.2007.12.001

Steroids

Volume 73, Issue 4, April 2008, Pages 417-423

https://doi.org/10.1016/j.steroids.2007.12.001 Get rights and content

Abstract

Conclusive in vivo evidence regarding the enzyme responsible for steroid hormone 5β-reduction has not been obtained, although studies have suggested it may be the same enzyme as that utilized for cholic acid and chenodeoxycholic bile-acid synthesis. We have recorded the steroid metabolome of a patient with a defect in the “bile-acid” 5β-reductase (AKR1D1) and from this confirm that this enzyme is additionally responsible for steroid hormone metabolism. The 13-year old patient has been investigated since infancy because of a cholestasis phenotype caused by bile-acid insufficiency. Several years ago it was shown that she had a 662C > T missense mutation in AKR1D1 causing a Pro198Leu substitution. It was found that the patient had an almost total absence of 5β-reduced metabolites of corticosteroids and severely reduced production of 5β-reduced metabolites of other steroids. The patient is healthy in spite of her earlier hepatic failure and is on no treatment. All her vital signs were normal, as were results of many biochemical analyses. She had normal pubertal changes and experiences regular menstrual cycles. There was no evidence for any clinical condition that could be attributed to attenuated ability to metabolize steroids in normal fashion. Both parents were heterozygous for the mutation but the steroid excretion was entirely normal, although an older female sibling showed definitive evidence for attenuated 5β-reduction of cortisol. A younger brother had a normal steroid metabolome. The sibling genotypes were not available.

Introduction

Reduction of sterols and steroids with a 3-oxo-4-ene structure is a common transformation. This is a two-step procedure, 5α- or 5β-reduction occurring first followed by the action of 3α-hydroxysteroid dehydrogenase. Reduction at 5α- has been most extensively studied following the finding that this reaction was involved in generation of the hormone 5α-dihydrotestosterone (5α-DHT), while comparatively little attention has been paid to 5β-reduction. However, this reaction has vital synthetic roles as an integral part of bile-acid synthesis, and a catabolic role in the degradative metabolism of active steroid hormones, about 2/3 of the mass of which are deactivated by this mechanism prior to excretion.

The human bile-acid biosynthetic enzyme has been cloned and is now known as AKR1D1. It has been expressed in COS cells and shows high 5β-reductase activity to bile-acid intermediates but low or absent activity to steroid hormones [1]. This suggests that may be more than one enzyme is involved in human 5β-reduction, a question that could be resolved by studying the urinary steroid metabolome of a human “knockout” of the enzyme, and over a period of 20 years there have been several reports of patients with putative 5β-reductase deficiency [2], [3], [4], [5], [6]. This disorder (congenital bile-acid synthesis defect type 2 (CBAS2), OMIM:235555) is known for a cholestasis and liver failure phenotype, which can be fatal in infancy. Lemonde et al. found inactivating AKR1D1 mutations in three patients with the disorder [7]. Typically the treatment for these patients is administration of exogenous bile-acids or liver transplantation. However, 11 years after publication of a case report on one of these individuals [3], [7], the female patient is thriving and has apparently no current need for bile-acid supplementation, possibly as a result of active formation of “replacement” 5α- (or “allo”) bile-acids. Daugherty et al. [8] have also studied patients with the disorder and have noted near-normalization of liver function with age and/or oral bile-acid administration.

We have revisited this case to determine if steroid (as distinct from sterol) 5β-reduction was affected, and evaluate the impact of the disorder on steroid hormone homeostasis, particularly related to clinical problems of an endocrine nature.

Section snippets

Patient

The patient is from the Italian province of Sardinia, the second child of parents not known to be consanguineous. Sardinia is a Mediterranean island with much of the population relatively isolated from mainland European communities for centuries. The patient's ancestors are autochthonous and the parents are both from the north-west region of the island. The patient presented at 3 weeks of age with cholestasis associated with steatorrhoea, failure to thrive and rickets. A liver biopsy at 3

Results

The excretion of corticosteroids (cortisol and corticosterone metabolites) by the patient and family are given in Table 2. The absolute amounts (μg/24 h) are approximations since they were calculated by multiplying quantitative values we obtained (μg/ml) by average volume excretions of the age-matched controls utilized in our laboratory. The relative excretions of individual compounds are authentic. While values listed this way are not accurate they permit easy comparison of the excretions of

Discussion

The presence in urine of steroid metabolites reduced at 5β has been known since 1930s but almost all studies on the enzyme 5β-reductase have concerned its important role in bile-acid synthesis. The “bile-acid synthesis enzyme” has been shown to be cytosolic and utilizes NADPH as electron donor. Berseus in 1967 partially purified the enzyme from rat liver and determined that it was effective in the reduction of bile-acid precursors and all forms of steroid hormones containing the 3-oxo-4-ene

Acknowledgements

This work was supported by the Wellcome Trust program grant 066357 to PMS. Thanks to Catherine Chong for expert technical assistance.

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However, in some cases, liver function can continue to deteriorate necessitating liver transplantation [194]. Despite the potential severity of these cases, survival into adulthood in the absence of treatment is reported [188]. Although the utility of 5βR inhibition is yet to be justified and could be questioned bearing in mind the phenotype associated with mutations in 5βR, compounds have been developed and tested as selective inhibitors.
The 5-reductases (5α-reductase types 1, 2 and 3 [5αR1−3], 5β-reductase [5βR]) are steroid hormone metabolising enzymes that hold fundamental roles in human physiology and pathology. They possess broad substrate specificity converting many steroid hormones to their 5α- and 5β-reduced metabolites, as well as catalysing crucial steps in bile acid synthesis. 5αRs are fundamentally important in urogenital development by converting testosterone to the more potent androgen 5α-dihydrotestosterone (5αDHT); inactivating mutations in 5αR2 lead to disorders of sexual development. Due to the ability of the 5αRs to generate 5αDHT, they are an established drug target, and 5αR inhibitors are widely used for the treatment of androgen-dependent benign or malignant prostatic diseases.
There is an emerging body of evidence to suggest that the 5-reductases can impact upon aspects of health and disease (other than urogenital development); alterations in their expression and activity have been associated with metabolic disease, polycystic ovarian syndrome, inflammation and bone metabolism. This review will outline the evidence base for the extra-urogenital role of 5-reductases from in vitro cell systems, pre-clinical models and human studies, and highlight the potential adverse effects of 5αR inhibition in human health and disease.
Quantitative analysis of mRNA expression levels of aldo-keto reductase and short-chain dehydrogenase/reductase isoforms in human livers
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AKR1D1 is involved in the synthesis of primary bile acid from cholesterol, and its defect causes inborn errors of bile acid metabolism [20]. AKR1D1 catalyzes the reduction of steroid hormones such as testosterone and progesterone [21]. It has been reported that the AKR1D1 mRNA level is significantly higher in the livers from heterozygotes and homozygotes of SNP at the 3′-UTR region of AKR1D1 (g.45267C > T; rs1872930, global minor allele frequency: 22.2%) than that from wild-type homozygotes [22].
The aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies are responsible for the reduction in compounds containing the aldehyde, ketone, and quinone groups. In humans, 12 AKR isoforms (AKR1A1, AKR1B1, AKR1B10, AKR1B15, AKR1C1, AKR1C2, AKR1C3, AKR1C4, AKR1D1, AKR1E2, AKR7A2, and AKR7A3) and 6 SDR isoforms (CBR1, CBR3, CBR4, HSD11B1, DHRS4, and DCXR) have been found to catalyze the reduction in xenobiotics, but their hepatic expression levels are unclear. The purpose of this study is to determine the absolute mRNA expression levels of these 18 isoforms in the human liver. In 22 human livers, all isoforms, except for AKR1B15, are expressed, and AKR1C2 (on average 1.6 × 10⁶ copy/μg total RNA), AKR1C3 (1.3 × 10⁶), AKR1C1 (1.3 × 10⁶), CBR1 (9.7 × 10⁵), and HSD11B1 (1.1 × 10⁶) are abundant, representing 67% of the total expression of reductases in the liver. The expression levels of AKR1C2, AKR1C3, AKR1C1, CBR1, and HSD11B1 are significantly correlated with each other, except between AKR1C2 and CBR1, suggesting that they might be regulated by common factor(s). In conclusion, this study comprehensively determined the absolute expression of mRNA expression of each AKR and SDR isoform in the human liver.
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Advances in technology have allowed for the sensitive, specific, and simultaneous quantitative profiling of steroid precursors, bioactive steroids and inactive metabolites, facilitating comprehensive characterization of the serum and urine steroid metabolomes. The quantification of steroid panels is therefore gaining favor over quantification of single marker metabolites in the clinical and research laboratories. However, although the biochemical pathways for the biosynthesis and metabolism of steroid hormones are now well defined, a gulf still exists between this knowledge and its application to the measured steroid profiles. In this review, we present an overview of steroid hormone biosynthesis and metabolism by the liver and peripheral tissues, specifically highlighting the pathways linking and differentiating the serum and urine steroid metabolomes. A brief overview of the methodology used in steroid profiling is also provided.
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In this work, genetic manipulation of AKR1D1 expression and activity regulated the inflammatory response in human hepatoma cells by enhancing the expression and secretion of pro-inflammatory cytokines alongside activation of the NF-κB pathway. In line with this, a significant number of studies have linked cholestasis and consequent liver inflammation and dysfunction with mutations in AKR1D1 [64–66]. It is thought that these mutations lead to increased levels of bile acid precursors, allo-bile acids (bile acids produced via 5αR activity) and low levels of CDCA (due to 27-hydroxylase activity and induction of the alternative pathway), which can then drive liver injury.
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Human liver biopsies were obtained from 34 obese patients and AKR1D1 mRNA expression levels were measured using qPCR. Genetic manipulation of AKR1D1 was performed in human HepG2 and Huh7 liver cell lines. Metabolic assessments were made using transcriptome analysis, western blotting, mass spectrometry, clinical biochemistry, and enzyme immunoassays.
In human liver biopsies, AKR1D1 expression decreased with advancing steatosis, fibrosis and inflammation. Expression was decreased in patients with type 2 diabetes. In human liver cell lines, AKR1D1 knockdown decreased primary bile acid biosynthesis and steroid hormone clearance. RNA-sequencing identified disruption of key metabolic pathways, including insulin action and fatty acid metabolism. AKR1D1 knockdown increased hepatocyte triglyceride accumulation, insulin sensitivity, and glycogen synthesis, through increased de novo lipogenesis and decreased β-oxidation, fueling hepatocyte inflammation. Pharmacological manipulation of bile acid receptor activation prevented the induction of lipogenic and carbohydrate genes, suggesting that the observed metabolic phenotype is driven through bile acid rather than steroid hormone availability.
Genetic manipulation of AKR1D1 regulates the metabolic phenotype of human hepatoma cell lines, driving steatosis and inflammation. Taken together, the observation that AKR1D1 mRNA is down-regulated with advancing NAFLD suggests that it may have a crucial role in the pathogenesis and progression of the disease.
AKR1D1 regulates glucocorticoid availability and glucocorticoid receptor activation in human hepatoma cells
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Steroid hormones, including glucocorticoids and androgens, have potent actions to regulate many cellular processes within the liver. The steroid A-ring reductase, 5β-reductase (AKR1D1), is predominantly expressed in the liver, where it inactivates steroid hormones and, in addition, plays a crucial role in bile acid synthesis. However, the precise functional role of AKR1D1 to regulate steroid hormone action in vitro has not been demonstrated. We have therefore hypothesised that genetic manipulation of AKR1D1 has the potential to regulate glucocorticoid availability and action in human hepatocytes.
In both liver (HepG2) and non-liver cell (HEK293) lines, AKR1D1 over-expression increased glucocorticoid clearance with a concomitant decrease in the activation of the glucocorticoid receptor and the down-stream expression of glucocorticoid target genes. Conversely, knockdown of AKR1D1 using siRNA decreased glucocorticoid clearance and reduced the generation of 5β-reduced metabolites. In addition, the two 5α-reductase inhibitors finasteride and dutasteride failed to effectively inhibit AKR1D1 activity in either cell-free or hepatocellular systems.
Through manipulation of AKR1D1 expression and activity, we have demonstrated its potent ability to regulate glucocorticoid availability and receptor activation within human hepatoma cells. These data suggest that AKR1D1 may have an important role in regulating endogenous (and potentially exogenous) glucocorticoid action that may be of particular relevance to physiological and pathophysiological processes affecting the liver.

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^☆: Steroid metabolites without specified 5-hydrogen orientation are 3α-hydroxy-5β-steroids.

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Human Δ4-3-oxosteroid 5β-reductase (AKR1D1) deficiency and steroid metabolism☆

Abstract

Introduction

Section snippets

Patient

Results

Discussion

Acknowledgements

Lancet

Lancet

J Steroid Biochem Mol Biol

J Biol Chem

FEBS Lett

Biochim Biophys Acta

Cloning and expression of cDNA of human Δ4-3-oxosteroid 5β-reductase and substrate specificity of the expressed enzyme

Eur J Biochem

Δ4-3-Oxosteroid 5β-reductase deficiency: failure of ursodeoxycholic acid treatment and response to chenodeoxycholic acid plus cholic acid

Gut

Δ4-3-Oxosteroid 5β-reductase deficiency described in identical twins with neonatal hepatitis A new inborn error in bile acid synthesis

J Clin Investig

Human Δ⁴-3-oxosteroid 5β-reductase (AKR1D1) deficiency and steroid metabolism☆

Cloning and expression of cDNA of human Δ⁴-3-oxosteroid 5β-reductase and substrate specificity of the expressed enzyme

Δ⁴-3-Oxosteroid 5β-reductase deficiency: failure of ursodeoxycholic acid treatment and response to chenodeoxycholic acid plus cholic acid

Δ⁴-3-Oxosteroid 5β-reductase deficiency described in identical twins with neonatal hepatitis A new inborn error in bile acid synthesis