Two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD) interconvert active cortisol and inactive cortisone. 11β-HSD2 (renal) acts only as a dehydrogenase, converting cortisol to cortisone. 11β-HSD1 (liver) is a bi-directional enzyme in cell homogenates, whereas in intact cells it typically displays oxo-reductase activity, generating cortisol from cortisone. We recently established that cortisone reductase deficiency is a digenic disease requiring mutations in both the gene encoding 11β-HSD1 and in the gene for a novel enzyme located within the lumen of the endoplasmic reticulum (ER), hexose-6-phosphate dehydrogenase (H6PDH). This latter enzyme generates NADPH, the co-factor required for oxo-reductase activity. Therefore, we hypothesized that H6PDH expression may be an important determinant of 11β-HSD1 oxo-reductase activity. Transient transfection of chinese hamster ovary (CHO) cells with 11β-HSD1 resulted in the appearance of both oxo-reductase and dehydrogenase activities in intact cells. Co-transfection of 11β-HSD1 with H6PDH increased oxo-reductase activity whilst virtually eliminating dehydrogenase activity. In contrast, H6PDH had no effect on reaction direction of 11β-HSD2, nor did the cytosolic enzyme, glucose-6-phosphate dehydrogenase (G6PD) affect 11β-HSD1 oxo-reductase activity. Conversely in HEK 293 cells stably transfected with 11β-HSD1 cDNA, transfection of an H6PDH siRNA reduced 11β-HSD1 oxo-reductase activity whilst simultaneously increasing 11β-HSD1 dehydrogenase activity. In human omental preadipocytes obtained from 15 females of variable body mass index (BMI), H6PDH mRNA levels positively correlated with 11β-HSD1 oxo-reductase activity, independent of 11β-HSD1 mRNA levels. H6PDH expression increased 5.3-fold across adipocyte differentiation (P<0.05) and was associated with a switch from 11β-HSD1 dehydrogenase to oxo-reductase activity. In conclusion, H6PDH is a crucial determinant of 11β-HSD1 oxo-reductase activity in intact cells. Through its interaction with 11β-HSD1, H6PDH may represent a novel target in the pathogenesis and treatment of obesity.

Steroid hormones, including glucocorticoids and androgens, exert a wide variety of effects in the body across almost all tissues. The steroid A-ring 5β-reductase (AKR1D1) is expressed in human liver and testes, and three splice variants have been identified (AKR1D1-001, AKR1D1-002, AKR1D1-006). Amongst these, AKR1D1-002 is the best described; it modulates steroid hormone availability and catalyses an important step in bile acid biosynthesis. However, specific activity and expression of AKR1D1-001 and AKR1D1-006 are unknown. Expression of AKR1D1 variants were measured in human liver biopsies and hepatoma cell lines by qPCR. Their three-dimensional (3D) structures were predicted using in silico approaches. AKR1D1 variants were overexpressed in HEK293 cells, and successful overexpression confirmed by qPCR and Western blotting. Cells were treated with either cortisol, dexamethasone, prednisolone, testosterone or androstenedione, and steroid hormone clearance was measured by mass spectrometry. Glucocorticoid and androgen receptor activation were determined by luciferase reporter assays. AKR1D1-002 and AKR1D1-001 are expressed in human liver, and only AKR1D1-006 is expressed in human testes. Following overexpression, AKR1D1-001 and AKR1D1-006 protein levels were lower than AKR1D1-002, but significantly increased following treatment with the proteasomal inhibitor, MG-132. AKR1D1-002 efficiently metabolised glucocorticoids and androgens and decreased receptor activation. AKR1D1-001 and AKR1D1-006 poorly metabolised dexamethasone, but neither protein metabolised cortisol, prednisolone, testosterone or androstenedione. We have demonstrated the differential expression and role of AKR1D1 variants in steroid hormone clearance and receptor activation in vitro. AKR1D1-002 is the predominant functional protein in steroidogenic and metabolic tissues. In addition, AKR1D1-001 and AKR1D1-006 may have a limited, steroid-specific role in the regulation of dexamethasone action.