Elucidate the role of 5β-reductase (AKR1D1) in oxysterol signalling in human hepatocytes

Nikolaos Nikolaou, University of Oxford

 

Aims

5β-reductase (AKR1D1) is an enzyme highly expressed in human liver, where it catalyses a fundamental step in bile acid (BA) synthesis. BAs as well as their intermediary products (oxysterols) have been implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and liver cancer. My recent findings have demonstrated decreased AKR1D1 expression in NAFLD patients (Nikolaou et al. 2019).

Expanding these observations, I have now revealed that genetic manipulation of AKR1D1 expression, using siRNA approaches, regulates cell proliferation and cell cycle in liver cancer cells. I hypothesise that the observed effects on proliferative phenotype are the results of alterations in BA and oxysterol composition, caused by AKR1D1 knockdown.

With this grant, I would like to visit the laboratory of Professor Carolyn Cummins at the University of Toronto, Canada. Professor Cummins is a leading expert in oxysterol metabolome analysis and her group have established methodologies for the measurement of oxysterol concentrations in biological samples.

My aims are:

1. Analyse the oxysterol metabolome profile of human hepatoma cells, following genetic manipulation of AKR1D1 expression
2. Analyse the oxysterol metabolome profile of human hepatoma cells and primary human hepatocytes, following treatment with novel pharmaceutical inhibitors that have the potential to block AKR1D1 activity

 

Evaluation

I would like to thank the Bioscientifica Trust for this award and the opportunity I was given to visit the laboratory of Prof. Carolyn Cummins at the University of Toronto, Canada. The visit undoubtedly enhanced my research on the role of AKR1D1 in hepatic oxysterol signalling, and both aims addressed in my application were successfully achieved. In addition, it helped me expose myself and gain experience in new analytical methods (LC-MS/MS), as well as further explore the role of nuclear receptors in metabolic liver disease.

During my time in Prof. Cummins’ lab, I successfully develop a novel protocol to extract, detect and quantify intracellular and secreted oxysterols from cell culture samples, including cell pellets and conditioned cell media samples. The results confirmed my research hypothesis regarding the ability of AKR1D1 to regulate oxysterol synthesis, and the generated data are going to be included in the upcoming manuscript, which we plan to submit within the following year. In parallel, I presented my current research in two 1-hour seminars, followed by 15min for questions and fruitful discussion.

Furthermore, I was given the opportunity to perform additional experiments for a second research manuscript regarding potential differences in oxysterol concentrations in mouse liver samples, following genetic manipulation of the mouse Akr1d1. The manuscript has already been submitted and is currently under review, and the Trust’s contribution has been acknowledged in the relevant funding section.

Crucially, I was able to analyse a third set of cell culture samples, and these results will be now used as preliminary data for my upcoming personal immediate post-doctoral applications.

 

Grant awarded: £2,000