In situ glucosylceramide synthesis and its pharmacological inhibition analysed in cells by ¹³C₅‐sphingosine precursor feeding and mass spectrometry

Abstract

Edited by Sandro SonninoGlycosphingolipids (GSLs) fulfil diverse functions in cells. Abnormalities in their metabolism are associated with specific pathologies and, consequently, the pharmacological modulation of GSLs is considered a therapeutic avenue. The accurate measurement of in situ metabolism of GSLs and the modulatory impact of drugs is warranted. Employing synthesised sphingosine and sphinganine containing 13 C atoms, we developed a method to monitor the de novo synthesis of glucosylceramide, the precursor of complex GSLs, by the enzyme glucosylceramide synthase (GCS). We show that feeding cells with isotope-labelled precursor combined with liquid chromatography-mass spectrometry (MS)/MS analysis allows accurate determination of the IC 50 values of therapeutically considered inhibitors (iminosugars and ceramide mimics) of GCS in cultured cells. Acquired data were comparable to those obtained with an earlier method using artificial fluorescently labelled ceramide to feed cells.

Unexpected phenotypic and molecular changes of combined glucocerebrosidase and acid sphingomyelinase deficiency

“…Future work should involve metabolic analyses of sphingolipids separated by cell type and by specific cellular fractions to produce a spatial understanding of the distinct sphingolipid metabolism and distribution across the different gba1 −/− and smpd1 −/− genotypes. Furthermore, whole-body analyses using recently developed techniques to monitor in situ glucosylceramide generation would give novel insights into the glycolipid dysregulation in our double mutants ( Katzy et al, 2022 ).…”