Endocrine Abstracts

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic disease. 5β-reductase (AKR1D1) is highly expressed in human liver where it inactivates steroid hormones and catalyzes a fundamental step in bile acid synthesis. Steroid hormones, including glucocorticoids, as well as bile acids are established regulators of metabolic phenotype. We hypothesized that AKR1D1 plays a crucial regulatory role in hepatic metabolic homeostasis. Genetic manipulation of AKR1D1 (over-expression, siRNA knockdown) was performed in human liver HepG2 and Huh7 cells. Gene expression changes were confirmed by qPCR. Functional activity, assessed using gas chromatography mass spectrometry to measure cortisone clearance and tetrahydrocortisone generation was paralleled by the anticipated changes in glucocorticoid receptor activation measured by luciferase reporter assays. AKR1D1 knockdown in HepG2 cells increased glucose transporter mRNA expression and extracellular glucose concentrations in the cell media decreased (15.3±1.5 vs 12.1±0.9 μmol/mg, P<0.05) while intracellular glycogen levels were increased (18.9±0.3 vs 22.7±0.3 μg/ml, P<0.05). AKR1D1 knockdown increased Fatty Acid Synthase and Acetyl CoA Carboxylase 1 expression, the rate-limiting step in de novo lipogenesis, DNL) (0.52±0.06 vs 0.89±0.04, P<0.01), and increased intracellular triglyceride (54.3±12.7 vs 73.3±11.0 nmol/mg, P<0.01). Furthermore, 3-hydroxybutyrate levels in the cell media were reduced, indicative of impaired fatty acid oxidation (18.7±2.3 vs 11.4±2.7 nmol/mg, P<0.01). Mass spectrometry analysis of lipid composition demonstrated increased palmitic and palmitoleic acid production consistent with increased DNL and fatty acid saturation. In addition, bile acid composition was altered with a significant increase in chenodeoxycholic acid levels. Conversely, pharmacological manipulation of the bile acid receptors FXR and LXR using the FXR agonist GW4064 and LXR antagonist 22-S-Hydroxycholesterol rescued HepG2 cells from metabolic dysfunction by reducing the expression of lipogenic genes. Furthermore, AKR1D1 knockdown increased proinflammatory cytokine IL-1, IL-6 and IL-8 mRNA expression; changes were confirmed by elevated cell media IL-8 levels (4.68±0.70 vs 13.39±2.28 ng/ml, P<0.05), increased IκBα degradation and induced IRE-1α protein expression, indicative of inflammation and cellular ER stress. In conclusion, AKR1D1 activity regulates steroid hormone and bile availability, potently modulating hepatic carbohydrate and lipid metabolism in addition to an inflammatory phenotype suggesting a crucial role in the pathophysiology of NAFLD.