Effect of gender genotype on sphingolipid metabolism: a study in C57BL/6J and PCSK9-KO mice

Background and Aim: Sphingolipids, a lipid class which includes ceramides as the central sphingolipid species, are constituents of cell membranes and circulate in plasma lipoproteins. Alterations of plasma ceramide levels or composition have been associated with an increased incidence of dysmetabolic conditions and atherosclerotic cardiovascular disease. This study was aimed at investigating, in mouse models, whether Pcsk9 deletion was able to affect ceramide homeostasis. Methods: C57BL/6J mice and Proprotein convertase subtilisin/ kexin 9 gene knock-out (Pcsk9-KO) mice in the C57BL/6J background were enrolled. From 6 to 22 weeks of age, mice were fed either a standard rodent diet (SD) containing 3% fat or a Western-type diet (WD) containing 0.2% cholesterol and 21% fat. Each experimental group was composed of both males (n=3-8) and females (n=3-8).At the end of the experimental period, blood was collected from fasted mice. Mice were then sacrificed, and the livers were harvested to evaluate the expression of genes involved in ceramide synthesis/metabolism by quantitative PCR (qPCR). Lipidomic analysis was performed on plasma and liver samples. Results: On SD, plasma levels of 18:1 ceramides and glycosphingolipids (GlcGalCer, LacCer) were higher in C57Bl/6J mice compared with Pcsk9-KO mice of both sexes. In both genotypes, and particularly in male mice, WD caused an increase of ceramide plasma levels, which however remained lower in Pcsk9-KO compared to C57Bl/6J mice. Differently from plasma, sphingolipid levels in the liver were comparable between the two genotypes on SD and showed similar increases after WD. The evaluation of gender differences within each genotype showed that the levels of several sphingolipid classes (GlcGalCer, LacCer, Gb3, SM) in the liver were higher in C57Bl/6 females than in males, both on SD and WD. The expression of several genes encoding for enzymes involved in the ceramide biosynthetic pathways was analysed. The results obtained indicated that, for some of the genes, there was no shared expression profile among groups. Of note, a strong upregulation of the ceramide de novo synthesis was observed in WD-fed Pcsk9-KO females. Conclusions: In spite of a differently regulated hepatic expression of genes involved in ceramide homeostasis, sphingolipid levels in liver were similar between Pcsk9-KO and C57Bl/6J mice, when the same gender and dietary condition were compared. Further analyses will be carried out to extend the evaluation of gene expression to the biosynthesis of ceramide-derived glycosphingolipids, such as GlcGalCer and LacCer.