Investigating DHCR24 as a protector against cellular stress : more than just a cholesterol-synthesising enzyme

University of Technology Sydney. Faculty of Science.Atherosclerosis and insulin resistance are globally prevalent metabolic diseases, primarily driven by endothelial and hepatic inflammation, respectively. High density lipoprotein (HDL) reduces the inflammation in models of atherosclerosis and insulin sensitivity, and in doing so, improves these conditions. Our laboratory has demonstrated that in human coronary artery endothelial cells (HCAECs), HDL’s suppression of the inflammatory response is a gene-regulated process and that 3β-hydroxysteroid-Δ24 reductase (DHCR24) is one of the most upregulated genes by HDL. DHCR24 is a cholesterol biosynthesis enzyme however, recent work in various cell types shows DHCR24 emerging as a potent multifaceted protein protecting against cellular stress. This study commenced with confirming apolipoprotein I (apoA-I) rHDL increases DHCR24 expression in HCAECs, while revealing for the first time that HDL also increases DHCR24 expression in human hepatoma 7 (HuH7) cells. This study next questioned whether DHCR24 mimics apoA-I rHDL’s suppression of the inflammatory response in these cell types. The data presented provides proof-of-principle that DHCR24 replicates HDL’s suppression of tumour necrosis factor-alpha (TNF-α)-induced intracellular cell adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) levels in HCAECs, and interleukin 8 (IL-8) levels in HuH7 cells. Characterising DHCR24’s role was explored to learn more about how DHCR24 suppresses a TNF-α-induced inflammatory response in HCAECs and HuH7 cells. This study showed that DHCR24’s oxidoreductase region mediates DHCR24’s suppression of a TNF-α-induced inflammatory response in these cell types. This effect occurred without DHCR24 increasing cholesterol levels, despite the oxidoreductase site’s integral role in cholesterol biosynthesis. These are exciting results as they indicate that DHCR24 is more than just a cholesterol biosynthesis enzyme in HCAECs and HuH7 cells. Elucidating the mechanisms by which DHCR24 suppresses a TNF-α-induced inflammatory response in HCAECs and HuH7 cells highlighted a cell type-specific nature of DHCR24’s activity, which is in keeping with reports in the literature. In keeping with this cell type-specificity, a TNF-α-induced inflammatory response was suppressed, in part, by a decreased endoplasmic reticulum (ER) stress response in HCAECs. Interestingly, this did not occur in HuH7 cells. The work here suggests that this is attributed to the increased cholesterol content in HuH7 cells compared to HCAECs. DHCR24’s cell type-specific effects are reinforced by the cellular response of DHCR24 levels to TNF-α-activation – in HCAECs, DHCR24 levels were modestly increased; conversely in HuH7 cells, TNF-α-activation markedly decreased DHCR24 levels. Moreover, TNF-α-activation caused DHCR24’s translocation from its ER-localisation to the cytoplasm and nucleus, while in HuH7 cells, DHCR24 remained localised peri-nuclearly. This data provides novel mechanistic insight into DHCR24’s multifunctional role in two different cell types, laying the foundation for potential DHCR24-based therapeutics, and provides impetus for further investigation of DHCR24

The use of tandem yeast and mammalian cell in vitro androgen bioassays to detect androgens in internet-sourced sport supplements

Sport supplements containing steroids never approved for therapeutic use have the potential for abuse by athletes. Most are marketed online and may contain undisclosed steroids yet are readily available despite lacking toxicological or pharmacological evaluation. In this study, 18 supplements purchased online underwent organic solvent extraction to isolate any steroids they contained. From the 18 supplements, 19 steroids were identified and for each, its intrinsic androgenic potency was determined by a yeast cell (Saccharomyces cerevisiae) androgen bioassay and its potential androgenic potency was determined by a liver (HuH7) cell androgen bioassay. The yeast bioassay showed that of the 19 steroids tested, 6 demonstrated strong intrinsic bioactivity, with 4 metabolically activated to even stronger androgens. Moreover, 4 steroids with moderate and 1 with intrinsically weak androgenic bioactivity were activated to more potent androgens. Finally, 8 steroids were metabolically inactivated or deactivated into weaker androgens. Our results show that Internet-sourced sport supplements may contain intrinsically strong androgens, or precursors that can be metabolized to them. These potentially potent pharmacologically active steroids are being used without regulatory control or consumer awareness of their potential adverse effects