Sphingolipid de novo synthesis is upregulated in a macrophage model of Gaucher disease

Gaucher disease (GD) is an inborn error of sphingolipid metabolism characterised by a block in the lysosomal degradation of glucosylceramide (GlcCer), which consequently accumulates in the lysosomes of affected cells. The product of GlcCer degradation is ceramide, and it is reasonable to expect that there would be a reduction in ceramide in GD, but there is conflicting evidence in different cells and tissues. Using a stable isotope ¹³C₁₆-palmitate labelling method, we sought to investigate the impact of the block in GlcCer degradation on the sphingolipid metabolic pathway in a conduritol B epoxide (CBE)-induced GD macrophage model. Liquid chromatography-mass spectrometry was used to measure acyl-, base-, and dual-labelled sphingolipid 'heavy' ¹³C₁₆ isotopologues which were distinguished by their incorporation of ¹³C₁₆ into the acyl chain, sphingoid base, or both, respectively. Base- and dual-labelled C16:0 ceramide was consistently elevated in CBE-GD macrophages whereas base- and acyl-labelled C24:0 ceramide was unchanged or decreased, suggesting a preference for ¹³C₁₆-palmitate in the sphingolipid de novo pathway over acyl elongation. Confirmation of the ¹³C₁₆-palmitate labelling results was then assessed with the sphingolipid de novo intermediate, sphinganine (d17:0), which also showed elevation of base-labelled C16:0 ceramide in CBE-GD macrophages and no change in base-labelled C24:0 ceramide. As palmitate and sphinganine are both precursors of ceramide de novo synthesis, all base- and dual-labelled ceramides can be accurately assigned as strictly de novo-derived. Therefore, we show that stable isotope ¹³C₁₆-palmitate labelling can detect alterations in the sphingolipid pathway and conclude that de novo synthesis is upregulated in the CBE-GD macrophage model. This upregulation is likely a compensatory mechanism employed by the CBE-GD macrophage to maintain ceramide homeostasis, following the loss of its generation through GlcCer turnover.