Endocrine Abstracts

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of disease ranging from intrahepatic lipid accumulation to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Liver 5β-reductase (AKR1D1) catalyses a fundamental step in bile acid (BA) synthesis. BAs and BA intermediates are potent regulators of metabolic and proliferative phenotype. We have hypothesised that AKR1D1 plays a crucial role in NAFLD and HCC. Liver biopsies and serum samples were obtained from healthy subjects and patients with established NAFLD, cirrhosis and HCC. BA composition was determined by LC-MS. Genetic manipulation of AKR1D1 was performed in HepG2 cells. Effects on BA synthesis, lipid metabolism, cell cycle, proliferation and DNA damage were determined by LC-MS, qPCR, western blotting, flow cytometry, RNA-sequencing, biochemistry analysis, and comet assays. Serum BA and BA-intermediate levels were significantly elevated across NALFD severity, with a particular increase in the concentration of the AKR1D1 substrate 7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA). In line with this, AKR1D1 expression was significantly decreased in liver biopsies from patients with advancing steatosis, fibrosis, and HCC. In HepG2 cells, AKR1D1 knockdown increased 7-HOCA accumulation, and RNA-sequencing analysis identified dysregulated pathways impacting lipid metabolism, cell cycle and proliferation, consistent with increased triglyceride accumulation and impaired fatty acid oxidation. In addition, AKR1D1 knockdown induced DNA damage, downstream resulting in cell cycle arrest, impaired cell proliferation and enhanced apoptosis, suggesting a role for 7-HOCA in hepatocyte health. Confirming this, RNA-sequencing 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, alongside enhanced DNA damage. In conclusion, AKR1D1 decreases with NAFLD severity, resulting in increased 7-HOCA accumulation and downstream adverse effects on hepatic lipid metabolism and cell proliferation. Taken together, these data demonstrate the important role of 7-HOCA in NAFLD progression and reveal the potential of AKR1D1 manipulation for hepatoprotective therapies.