Endocrine Abstracts

Non-alcoholic fatty liver disease is the hepatic manifestation of the global epidemic of metabolic disease. It is tightly associated with obesity and type 2 diabetes, yet the precise mechanisms that drive its aetiology are not fully defined. Steroid hormones, including glucocorticoids and sex steroids, regulate metabolic phenotype, and in addition, bile acids have recently been identified as potent metabolic regulators. AKR1D1 (5β-reductase), is predominantly expressed in the liver, and is a crucial regulator of steroid hormone clearance as well as bile acid synthesis. Its role in pathogenesis of metabolic disease has not been examined. We have therefore developed systems to define the enzymology of human AKR1D1 in cell free assays and to determine the impact of manipulation of AKR1D1 expression and activity in human hepatocyte models. B21 Rosetta bacteria cells were transformed with an AKR1D1 construct (pNIC-CTHF+AKR1D1) and recombinant protein extracted and purified. A high throughput assay was developed to determine AKR1D1 activity, substrate specificity and enzyme kinetics. Furthermore, AKR1D1 activity was inhibited by Finasteride (selective 5αR2 inhibitor), but not Dutasteride (non-selective 5αR inhibitor). AKR1D1 mRNA expression was characterised in four different hepatoma cell lines (Hep3b, HepG2, C3A, and Huh7.0) as well as primary cultures of human hepatocytes. In addition, HepG2 cells were differentiated using an established protocol (including 1% DMSO treatment), and gene expression analyzed after 7, 14, and 21 days. Over-expression and siRNA knock down of AKR1D1 in HepG2 cells were also performed. AKR1D1 was highly expressed in human hepatoma cell lines and expression decreased across differentiation, to levels that were similar to those seen in primary cultures of human hepatocytes. Successful over-expression and knock down of AKR1D1 were confirmed in HepG2 cells using real-time PCR. Importantly, changes in gene expression were paralleled by functional activity as measured by progesterone clearance. We have characterised human AKR1D1 in cell-free systems and in established liver cell models. We have successfully manipulated AKR1D1 expression and activity that will serve as the platform for future studies to define its role in the regulation of metabolic phenotype within the liver.