Endocrine Abstracts

The hepatic enzyme 5β-reductase (AKR1D1) sits at the interface between two metabolic pathways, converting steroid hormones to their inactive 5β-reduced metabolites during steroid clearance, and as a step in the synthesis of bile acids from cholesterol. Both the steroid substrates and the bile acid products of AKR1D1 are potent hormones that regulate hepatic energy metabolism and inflammation. It is not known how these two functions are spatially organised within hepatocytes. The subcellular localisation of steroid metabolising enzymes is thought to have a role in determining their activity and substrate preference. Key bile acid synthesis enzymes upstream of AKR1D1 are localised to the endoplasmic reticulum (ER) membrane and we hypothesised that a fraction of AKR1D1 would localise at the ER to facilitate its role in bile acid synthesis. To test this hypothesis, AKR1D1 tagged with green fluorescent protein (AKR1D1-GFP), or GFP alone, were overexpressed in HepG2 hepatoma cells. The ER was labelled using a targeted red fluorescent protein (ER-RFP). Serial optical sections of cells expressing both AKR1D1-GFP (or GFP) and ER-RFP were imaged using Airyscan laser scanning confocal microscopy, and the 3D colocalisation of the fluorescent probes was quantified using ImageJ. There was no difference in the degree of colocalisation with ER-RFP between AKR1D1-GFP or GFP. These data suggest that AKR1D1 does not localise to the ER more than GFP alone. However, the overexpression of AKR1D1-GFP appeared to disrupt the normal localisation of AKR1D1 and so these results need to be confirmed in cells with endogenously tagged AKR1D1. AKR1D1-GFP was observed in both the cytoplasm and nucleus of HepG2 cells. To confirm whether native AKR1D1 localises to the nucleus, nuclear and cytoplasmic fractions were isolated from HepG2 cells and fraction purity confirmed by Western blot using Lamin A/C and β-Tubulin as controls. AKR1D1 was detected in the cytoplasmic but not the nuclear extract, suggesting that the nuclear staining may be an artefact of overexpression.