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 regulatory role in NAFLD and HCC. Human liver biopsies were obtained from 34 obese patients. Genetic manipulation of AKR1D1 (siRNA/shRNA) was performed in human HepG2 cells. Effects on BA synthesis, nuclear receptor activation, insulin sensitivity, cell cycle and proliferation were determined by LC-MS, qPCR, western blotting, flow cytometry, luciferase reporter assays, and RNA-sequencing. Recombinant human AKR1D1 was expressed in BL-21 bacteria cells, purified, and screened to identify novel AKR1D1 pharmacological inhibitors. In liver biopsies, AKR1D1 expression decreased with advancing steatosis, fibrosis, and inflammation. RNA-sequencing in AKR1D1-knockdown HepG2 cells identified dysregulated pathways impacting insulin signalling, DNA replication, cell cycle and proliferation. AKR1D1 knockdown decreased primary BA and increased AKR1D1-substrate (7α, hydroxy-cholestenone, 7α,12α, dihydroxy-cholestenone) concentrations, and increased insulin-stimulated AKT phosphorylation, consistent with enhanced insulin sensitivity. Additionally, AKR1D1 knockdown decreased cyclin-dependent kinase and increased cyclin-dependent kinase inhibitor expression, downstream resulting in cell cycle arrest at G1/S phase, impaired proliferation, and enhanced apoptosis. Complementing these findings, pharmacological inhibition of AKR1D1 using three novel AKR1D1 inhibitors (identified through a high-throughput drug screen of >300,000 compounds) impaired cell proliferation and proliferative gene expression. Luciferase assays revealed increased LXR activation following AKR1D1 knockdown, identifying the AKR1D1 substrates as novel endogenous LXR ligands. Pharmacological inhibition of LXR activation prevented the induction of metabolic and proliferative gene expression. In conclusion, AKR1D1 knockdown enhances insulin sensitivity, delays cell cycle, and inhibits proliferation through LXR-dependent mechanisms. Taken together, these data suggest a beneficial role of AKR1D1 inhibition in NAFLD and HCC.