AKR1D1 Human, His

What is AKR1D1 and what is its primary function in humans?

AKR1D1 is a member of the aldo-keto-reductase (AKR) superfamily 1 of enzymes and the first member of the 1D subfamily. The human gene consists of nine exons with six transcript variants identified, three of which lead to functional protein isoforms . AKR1D1 utilizes NADPH as a hydride donor and catalyzes a stereospecific irreversible double bond reduction between the C4 and C5 positions of the A-ring of steroids, creating an A/B cis-ring junction and introducing a 90° bend in the steroid structure . This structural alteration creates steroids with significantly different properties from either α,β-unsaturated or 5α-reduced steroids.

AKR1D1 serves two primary functions: inactivating steroid hormones (including glucocorticoids and androgens) to regulate their availability and receptor activation, and catalyzing a fundamental step in bile acid biosynthesis . We have recently demonstrated its potent ability to regulate glucocorticoid availability and receptor activation within human hepatoma cells, suggesting an important role in regulating both endogenous and potentially exogenous glucocorticoid action .

Where is AKR1D1 predominantly expressed in the human body?

AKR1D1 is principally expressed in the liver, where levels are more than ten-fold higher than in any other tissue . This high hepatic expression aligns with its dual roles in steroid hormone inactivation and bile acid synthesis, processes that are predominantly hepatic. AKR1D1 is also expressed in human testes, though with a different splice variant profile than observed in the liver .

The tissue-specific expression pattern of AKR1D1 splice variants is particularly noteworthy. AKR1D1-002 and AKR1D1-001 are expressed in human liver, while only AKR1D1-006 is expressed in human testes . This differential expression suggests tissue-specific roles in steroid hormone metabolism and may contribute to tissue-specific regulation of steroid hormone availability.

How does AKR1D1 metabolize different glucocorticoids?

AKR1D1 shows differential metabolism of endogenous versus synthetic glucocorticoids. In overexpression studies, cortisol (the endogenous glucocorticoid) was almost completely cleared within 24 hours in cells overexpressing AKR1D1 compared to controls . In contrast, synthetic glucocorticoids demonstrated significant resistance to AKR1D1-mediated metabolism, with only partial clearance of prednisolone and dexamethasone under the same conditions .

AKR1D1 converts cortisol to 5β-tetrahydrocortisol (5β-THF) and cortisone to 5β-tetrahydrocortisone (5β-THE) . This 5β-reduction significantly reduces glucocorticoid receptor activation by both increasing clearance of active glucocorticoids and converting them to metabolites with substantially reduced receptor binding affinity.

The three AKR1D1 splice variants also differ in their glucocorticoid metabolism capabilities. While AKR1D1-002 efficiently metabolizes both endogenous and synthetic glucocorticoids, AKR1D1-001 and AKR1D1-006 poorly metabolize dexamethasone and show negligible activity toward cortisol and prednisolone .

What role does AKR1D1 play in regulating gene expression?

AKR1D1 regulates gene expression primarily through its effect on nuclear hormone receptor activation. In dual transfection experiments with both AKR1D1 and a glucocorticoid-responsive element (GRE) luciferase construct, AKR1D1 overexpression decreased glucocorticoid receptor activation in the presence of cortisol . This reduced activation resulted in decreased mRNA expression of known glucocorticoid-regulated genes including SGK1, DUSP1, and GILZ .

The impact of AKR1D1 on gene expression extends beyond glucocorticoid-regulated pathways. AKR1D1 knockdown experiments revealed that the effects were mediated through multiple nuclear hormone receptors, including the glucocorticoid receptor (GR), pregnane X receptor (PXR), and farnesoid X receptor (FXR) . This multi-receptor effect altered the expression of genes involved in gluconeogenesis and glycogen synthesis .

Through its role in bile acid synthesis, AKR1D1 also indirectly regulates FXR-dependent gene expression, which impacts lipid metabolism and other metabolic pathways .

How is AKR1D1 itself regulated?

Glucocorticoids exert regulatory control over AKR1D1 expression and activity. Both in vitro and in vivo studies have demonstrated that dexamethasone treatment decreases AKR1D1 expression and activity . This creates a negative feedback loop that further limits glucocorticoid clearance and augments glucocorticoid action .

This negative regulation by glucocorticoids contrasts with their effect on 11β-HSD1 (which converts inactive cortisone to active cortisol), where glucocorticoids increase expression and activity . This differential regulation creates a scenario where glucocorticoid exposure simultaneously decreases inactivation (via AKR1D1) and increases activation (via 11β-HSD1), potentially exacerbating glucocorticoid excess.

At the protein level, the different AKR1D1 splice variants show differential regulation. AKR1D1-001 and AKR1D1-006 have lower protein levels than AKR1D1-002 when overexpressed, but their levels increase significantly following treatment with the proteasomal inhibitor MG-132 . This suggests that these variants are more susceptible to proteasomal degradation, representing an additional level of post-translational regulation.

What experimental approaches are most effective for studying AKR1D1 activity in vitro?

Multiple complementary approaches have proven effective for studying AKR1D1 in vitro:

• Overexpression systems: Transfection of human cell lines (HEK293, HepG2, Huh7) with AKR1D1-containing vectors has been successfully used to assess AKR1D1 activity . This approach allows for comparison of different splice variants and assessment of dose-dependent effects.

• siRNA-mediated knockdown: siRNA molecules (such as HSS1101183 and HSS1101184) have achieved >90% reduction in AKR1D1 mRNA expression in hepatoma cells . The protocol typically involves 20 nmol of AKR1D1 siRNA diluted in 25 μL OPTIMEM serum-free media combined with 2.5 μL Lipofectamine RNAiMAX diluted in 25 μL OPTIMEM, with 48-hour incubation prior to experimental treatments .

• Activity assays: Treatment of cells with steroid hormones followed by measurement of steroid clearance and metabolite formation using mass spectrometry provides direct assessment of AKR1D1 activity . This approach can measure the differential metabolism of various glucocorticoids and other steroids.

• Reporter assays: Dual transfection with both AKR1D1 and reporter constructs (e.g., GRE-luciferase) allows simultaneous manipulation of AKR1D1 and assessment of downstream signaling effects . This approach has been used to demonstrate AKR1D1's impact on glucocorticoid receptor activation.

• Western blotting: Protein detection using commercially available antibodies (e.g., HPA057002, Atlas Antibodies AB at 1/250 dilution) provides quantification of AKR1D1 protein levels . This technique is essential for verifying successful manipulation of AKR1D1 expression.

How do the different AKR1D1 splice variants impact steroid metabolism?

The three functional AKR1D1 splice variants demonstrate significant differences in their impact on steroid metabolism:

AKR1D1-002 is the predominant functional protein in steroidogenic and metabolic tissues . It efficiently metabolizes both glucocorticoids and androgens, significantly decreasing receptor activation for these hormones . When overexpressed, AKR1D1-002 shows robust protein expression and effectively reduces expression of glucocorticoid-regulated genes like SGK1, DUSP1, and GILZ .

AKR1D1-001 shows lower protein levels than AKR1D1-002 when overexpressed, suggesting greater susceptibility to degradation . Its protein levels increase significantly following treatment with the proteasomal inhibitor MG-132 . Functionally, AKR1D1-001 poorly metabolizes dexamethasone but does not effectively metabolize cortisol, prednisolone, testosterone, or androstenedione .

AKR1D1-006 shares similar characteristics with AKR1D1-001, showing lower protein levels that increase with proteasomal inhibition . It also poorly metabolizes dexamethasone but has negligible activity toward other tested steroids . Its unique expression in testes suggests a potential tissue-specific role in steroid metabolism .

The differential expression patterns and activities of these variants indicate specialized roles in different tissues, with AKR1D1-002 likely being the primary driver of hepatic steroid clearance.

What evidence exists for sexually dimorphic effects of AKR1D1?

These sexually dimorphic effects were associated with sex-specific changes in bile acid metabolism and composition, but without overt effects on glucocorticoid action . This suggests that the metabolic effects were primarily mediated through altered bile acid homeostasis rather than changes in glucocorticoid signaling.

Interestingly, male knockout mice were not protected against diet-induced obesity and insulin resistance , indicating that the metabolic phenotype is context-dependent and may be influenced by environmental factors such as diet.

How does AKR1D1 influence nuclear hormone receptor signaling?

AKR1D1 activity influences multiple nuclear hormone receptor signaling pathways:

For the Glucocorticoid Receptor (GR), AKR1D1 reduces GR activation by metabolizing glucocorticoids . AKR1D1 knockdown enhances expression of GR target genes (SGK1, DUSP1, GILZ) and affects gluconeogenic and glycogen synthesis gene expression .

Regarding the Farnesoid X Receptor (FXR), AKR1D1 activity is essential for bile acid synthesis, and changes in AKR1D1 expression alter bile acid pool size and composition . This impacts FXR activation and its downstream metabolic effects. AKR1D1 has been shown to regulate lipid metabolism largely through its effects on FXR activation .

For the Pregnane X Receptor (PXR), AKR1D1 knockdown alters PXR activation, contributing to changes in metabolic gene expression profiles .

The effects of AKR1D1 on these nuclear receptors creates a complex regulatory network where decreased AKR1D1 can simultaneously increase GR activation (through reduced glucocorticoid clearance) and alter FXR activation (through changes in bile acid synthesis) . These combined effects likely explain the broader metabolic impact of AKR1D1 beyond just steroid hormone regulation.

What is the relationship between AKR1D1 and metabolic disease?

The relationship between AKR1D1 and metabolic disease involves several interconnected pathways:

AKR1D1 regulates glucocorticoid availability and receptor activation, with implications for glucose metabolism . AKR1D1 knockdown enhances expression of gluconeogenic and glycogen synthesis genes , and excessive glucocorticoid action is associated with insulin resistance.

Through its role in bile acid synthesis, AKR1D1 affects bile acid composition and FXR activation . This impacts lipid metabolism, with knockout mice showing sex-specific changes in insulin sensitivity and lipid homeostasis .

Male Akr1d1-/- mice at 30 weeks of age show a complex metabolic phenotype with increased insulin sensitivity and reduced hepatic and adipose lipid accumulation, but also hypertriglyceridemia and increased intramuscular lipid deposition . The phenotype is sexually dimorphic, affecting only male mice .

In humans, AKR1D1's dual role in regulating both glucocorticoid and bile acid signaling suggests it could potentially contribute to conditions such as non-alcoholic fatty liver disease, insulin resistance, and dyslipidemia. The down-regulation of AKR1D1 by synthetic glucocorticoids may also contribute to the adverse metabolic effects of glucocorticoid therapy .

How can AKR1D1 activity be measured in human samples?

AKR1D1 activity can be measured in human samples through several complementary approaches:

Urinary steroid metabolite profiling using gas chromatography-mass spectrometry (GC-MS) can measure 5β-reduced metabolites, including 5β-tetrahydrocortisol (5β-THF) and 5β-tetrahydrocortisone (5β-THE) . This approach has been used to assess AKR1D1 activity in vivo in response to dexamethasone treatment .

Direct measurement of AKR1D1 mRNA expression in liver biopsies using qPCR and protein quantification by Western blotting can assess expression levels . This allows assessment of different splice variant expression patterns in human tissues.

In ex vivo systems, primary human hepatocytes can be used for activity assessments by treating with steroid substrates followed by measurement of 5β-reduced metabolites . This enables assessment of both endogenous AKR1D1 activity and response to various treatments.

Plasma bile acid profiling using liquid chromatography-mass spectrometry (LC-MS) can analyze bile acid species and intermediates that reflect AKR1D1 activity . Changes in bile acid composition can serve as an indirect marker of altered AKR1D1 function.

What are the implications of AKR1D1's differential metabolism of synthetic glucocorticoids?

The differential metabolism of synthetic glucocorticoids by AKR1D1 has several important implications:

Synthetic glucocorticoids like prednisolone and dexamethasone are cleared less efficiently by AKR1D1 compared to endogenous cortisol . This contributes to their extended half-lives and enhanced potency in clinical settings.

Dexamethasone not only resists AKR1D1 metabolism but also decreases AKR1D1 expression and activity both in vitro and in vivo . This creates a compounding effect where the synthetic glucocorticoid both resists clearance and reduces the expression of the enzyme responsible for its metabolism.

The AKR1D1 splice variants show differential metabolism of synthetic glucocorticoids, with AKR1D1-001 and AKR1D1-006 able to metabolize dexamethasone (albeit poorly) but not prednisolone . This suggests potential splice variant-specific effects on drug metabolism.

The reduced clearance and negative regulation of AKR1D1 by synthetic glucocorticoids may contribute to their adverse metabolic effects by creating a vicious cycle of enhanced glucocorticoid action . This has implications for understanding and potentially mitigating the metabolic side effects of glucocorticoid therapy.

Research suggests that the reduced clearance of synthetic glucocorticoids should be considered when evaluating their therapeutic window and potential for adverse effects, particularly in metabolically vulnerable populations.