Endocrine Abstracts

Introduction: Sphingosine-1-phosphate lyase (SGPL1) catalyses the final step in sphingolipid metabolism, irreversibly degrading the lipid signalling molecule sphingosine-1-phosphate (S1P). The relative abundance of S1P compared to its precursors sphingosine and ceramide finely tunes signal transduction for a wide range of cellular pathways including proliferation, apoptosis, migration and calcium handling. Loss-of-function mutations in SGPL1 cause a spectrum of disorders, including steroid-resistant nephrotic syndrome, primary adrenal insufficiency, ichthyosis and more. In each case, it is unclear how altered SGPL1 activity causes the phenotypes.

Methods: We generated isogenic SGPL1-knockout and stable SGPL1-overexpressing H295R adrenocortical cells to better analyse the role of SGPL1 in the adrenal gland. To investigate the effect of these perturbations on cell signalling and function we conducted MTT, circle scratch and seahorse XF assays, plus RNAseq, with western blotting and qPCR validation.

Results: We noticed a marked increase in proliferation in the overexpressing cells and a concordant decrease in proliferation in knockout cells. Similarly, overexpressing cells migrated faster while knockout cells were slower. We then sought transcriptome changes accompanying these phenotypic differences. Gene-set enrichment analysis of RNAseq data revealed a highly significant enrichment of genes associated with oxidative phosphorylation in the overexpressing cells, and a downregulation of genes involved in steroidogenesis in the knockout. Seahorse analysis revealed SGPL1 overexpressing H295R cells had increased basal and maximal respiration. SGPL1 knockout cells had markedly reduced non-mitochondrial oxygen consumption, which led us to believe there may be a difference in glycolysis in these cells. Surprisingly, we found the greatest differences in the overexpressing cells, which showed increased basal and maximal glycolysis, while the knockouts had a slightly reduced maximal glycolytic capacity.

Conclusions: SGPL1 expression correlates with growth and migration rates in H295R cells, with knockout reducing steroidogenic capacity and overexpression increasing metabolism.