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, in parallel, catalyzes a fundamental step in bile acid synthesis. Steroid hormones, including glucocorticoids, as well as bile acids (BAs) are established regulators of metabolic phenotype. We have 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, as measured by luciferase reporter assays. In addition, total BA production was decreased, resulting in disturbed BA composition. RNA sequencing analysis following AKR1D1 knockdown defined discrete dysregulated metabolic pathways, notably those impacting upon insulin action and fatty acid storage and utilization. Insulin sensitivity was enhanced with increased insulin-stimulated phosphorylation of AKT, mTOR and GSK-3β following AKR1D1 knockdown. Endorsing our cellular observations, hepatic total AKT levels in 12-week AKR1D1 knockout male mice were higher than in wild-type controls, with evidence of increased phosphorylation upon insulin stimulation. In vitro, AKR1D1 knockdown increased glucose transporter mRNA expression with an associated decrease in extracellular glucose concentrations (15.3±1.5 vs 12.1±0.9 mmol/mg, P<0.05) and increased intracellular glycogen accumulation (18.9±0.3 vs 22.7±0.3 ug/ml, P<0.05). In addition, Fatty Acid Synthase (FASN) and Acetyl CoA Carboxylase 1 (ACC1) expression, the rate-limiting step in de novo lipogenesis, DNL), were increased, resulting in enhanced phosphorylation of ACC and increased intracellular triglyceride accumulation (54.3±12.7 vs 73.3±11.0 nmol/mg, P<0.01). Mass spectrometry analysis of lipid composition demonstrated increased palmitic and palmitoleic acid synthesis, consistent with increased DNL and fatty acid saturation. Cell media 3-hydroxybutyrate levels were reduced (18.7±2.3 vs 11.4±2.7 nmol/mg, P<0.01), indicative of impaired fatty acid oxidation. Pharmacological manipulation of BA receptor activation, using the FXR agonist GW4064 and LXR antagonist 22(S)-Hydroxycholesterol, prevented the induction of lipogenic genes, suggesting that the observed metabolic phenotype is likely to be driven through BA rather than steroid hormone availability. In conclusion, AKR1D1 has the ability to regulate hepatocyte insulin sensitivity as well as carbohydrate and lipid metabolism, and therefore may have an as yet unexplored role in the pathophysiology of NAFLD.