Endocrine Abstracts

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic disease. Steroid hormones and bile acids are established regulators of metabolic phenotype. 5β-reductase (AKR1D1) is highly expressed in human liver where it inactivates steroid hormones and catalyzes a fundamental step in bile acid synthesis. We hypothesized that AKR1D1 plays a key role in hepatic metabolic homeostasis. Genetic manipulation of AKR1D1 was performed in human liver HepG2 and Huh7 cells. Gene expression changes were confirmed by qPCR, glucocorticoid clearance and 5β-metabolites generation, using gas chromatography-mass spectrometry. AKR1D1 over-expression decreased glucocorticoid receptor activation, as measured by luciferase reporter assays. RNAseq analysis in AKR1D1 knocked down HepG2 cells identified dysregulated pathways impacting upon metabolic pathways, insulin action and fatty acid metabolism. AKR1D1 knockdown increased glucose transporter mRNA expression and decreased cell media glucose concentrations, while increased intracellular glycogen levels. AKR1D1 knockdown increased FAS, SCD1, SREBP-1c and ACC1 expression, the rate-limiting step in de novo lipogenesis (DNL), and intracellular triglyceride, while cell media 3-hydroxybutyrate levels were reduced, indicative of impaired fatty acid oxidation. Mass spectrometry analysis of lipid composition demonstrated increased palmitic and palmitoleic acid production, consistent with increased DNL and fatty acid saturation. Furthermore, bile acid composition was altered with significantly increased chenodeoxycholic acid levels. Conversely, pharmacological manipulation of the bile acid receptors FXR and LXR using GW4064 (FXR agonist) and 22(S)-Hydroxycholesterol (LXR antagonist) rescued HepG2 cells from metabolic dysfunction by reducing lipogenic gene expression. AKR1D1 knockdown increased proinflammatory cytokine IL-1β, IL-6 and IL-8 mRNA expression; changes were confirmed by elevated cell media IL-6 and IL-8 levels, increased IκBα degradation and induced IRE1α protein expression, indicative of inflammation and ER stress. In conclusion, AKR1D1 activity regulates steroid hormone and bile acid availability, potently modulating hepatic carbohydrate and lipid metabolism, in addition to an inflammatory phenotype, suggesting a crucial role in NAFLD pathophysiology.