ECE2016 Guided Posters Receptors & Signalling (10 abstracts)
5β-reductase (AKR1D1) is a regulator of glucose homeostasis in human hepatocytes and development of model systems to define its role in metabolic liver disease
Nikolaou Nikolaos 1 , James Dunford 1 , Charlotte Green 1 , Wenhwa Lee 1 , Reina Lim 2 , Laura Gathercole 1 , Jane McKeating 2 , Udo Oppermann 1 , Leanne Hodson 1 & Jeremy Tomlinson 1
1University of Oxford, Oxford, UK; 2University of Birmingham, Birmingham, UK.
Non-alcoholic fatty liver disease is the hepatic manifestation of the global epidemic of metabolic disease. Steroid hormones, including glucocorticoids and sex steroids, regulate metabolic phenotype, and in addition, bile acids have recently been identified as potent metabolic regulators. 5β-reductase (AKR1D1) 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, to determine the impact of manipulation of AKR1D1 expression and activity in human hepatocyte models and we propose that AKR1D1 regulates glucose flux in human liver. B21 bacteria cells were transformed with an AKR1D1 construct and recombinant protein extracted and purified. A high-throughput assay was developed to determine AKR1D1 activity, substrate specificity and enzyme kinetics. AKR1D1 activity was inhibited by Finasteride (selective 5αR2 inhibitor), but not Dutasteride (non-selective 5αR inhibitor). AKR1D1 mRNA expression was characterized in four different hepatoma cell lines (Hep3b, HepG2, C3A and Huh7.0) as well as primary cultures of human hepatocytes. Over-expression and siRNA knockdown of AKR1D1 in HepG2 cells were performed and confirmed using qPCR. Changes in gene expression were paralleled by functional activity as measured by cortisone clearance and tetrahydrocortisone generation using GC/MS. AKR1D1 knockdown increased the mRNA expression of the glucose transporters GLUT1 and GLUT9, as measured by qPCR (GLUT1: 0.6±0.1 vs 1.71±0.1, P<0.05; GLUT9: 0.58±0.05 vs 1.35±0.08, P<0.05). In addition, AKR1D1 knockdown also increased the extracellular glucose concentration in the cell culture media (3.7±0.33 vs 5.36±0.5 umol/mg, P<0.05) with evidence of enhanced gluconeogenesis through increased PEPCK mRNA expression (0.13±0.03 vs 0.36±0.01AU, P<0.05). AKR1D1 over-expression did not impact upon cellular metabolic phenotype. We have characterised human AKR1D1 in cell-free systems and in established liver cell models. Furthermore, we have successfully manipulated AKR1D1 expression and activity and identified it as a potent regulator of glucose homeostasis within human hepatocytes.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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