Endocrine Abstracts

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of disease ranging from simple intrahepatic lipid accumulation to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). 5β-reductase (AKR1D1) is a liver enzyme that catalyses a fundamental step in bile acid (BA) synthesis. Both BAs and BA intermediates are established as potent regulators of metabolic and proliferative phenotype. We have hypothesised that AKR1D1 plays a crucial role in NAFLD and HCC. Human liver biopsies and serum samples were obtained from healthy subjects and patients with established NAFLD, cirrhosis and HCC. Alterations in BA synthesis and composition were determined by LC-MS. Genetic manipulation of AKR1D1 (siRNA/shRNA) was performed in human hepatoma HepG2 cells. Effects on BA synthesis, fatty acid metabolism, cell cycle, proliferation and DNA damage were determined by LC-MS, qPCR, western blotting, flow cytometry, RNA-sequencing, ILab biochemistry analyser, and single cell gel electrophoresis (comet assay). Total serum BA and BA intermediate levels were significantly elevated across NALFD disease severity, with a particular increase in the concentration of the AKR1D1 substrate 7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA) [control: 182.1±14.9 (n=19) vs HCC: 350.6±37.4 nM (n=20), P=1e-4). In line with this, AKR1D1 expression was significantly decreased in liver biopsies from patients with advancing steatosis, fibrosis, inflammation and HCC. In HepG2 cells, AKR1D1 knockdown decreased primary BA and increased 7-HOCA concentrations. RNA-sequencing analysis in AKR1D1-knockdown cells identified dysregulated pathways impacting lipid metabolism, cell cycle and proliferation, consistent with increased intracellular triglyceride accumulation and decreased fatty acid oxidation. In addition, AKR1D1 knockdown induced DNA damage, downstream resulting in cell cycle arrest at G1/S phase, impaired cell proliferation and enhanced apoptosis, suggesting a role for 7-HOCA in liver cell health. Confirming this, RNA-sequencing and subsequent pathway enrichment analysis in wild-type, 7-HOCA-treated HepG2 cells revealed a transcriptional profile similar to the one observed following AKR1D1 knockdown, with increased lipid and decreased proliferative gene expression, accompanied by enhanced DNA damage. Complementing these findings, pharmacological inhibition of AKR1D1 using three novel AKR1D1 inhibitors (identified through a high-throughput drug screen of >300,000 compounds) similarly impaired proliferative gene expression and cell proliferation. In conclusion, AKR1D1 expression is decreased in patients with NAFLD and HCC, and results in increased accumulation of its substrate, 7-HOCA, with downstream detrimental effects on hepatic triglyceride metabolism, fatty acid oxidation and cell proliferation. Taken together, these data demonstrate the crucial role of 7-HOCA in NAFLD/HCC progression and reveal the potential of AKR1D1 manipulation for hepatoprotective therapies.