ABSTRACTBackgroundDihydrosphingolipids are lipid molecules biosynthetically related to ceramides. An increase in ceramides is associated with enhanced fat storage in the liver and inhibition of their synthesis is reported to prevent the appearance of steatosis in animal models. However, the precise association of dihydrosphingolipids with non-alcoholic fatty liver disease (NAFLD) is yet to be established. We employed a diet-induced NAFLD mouse model to study the association between this class of compounds and disease progression.MethodsMice were fed a high-fat diet enriched in cholesterol and supplemented with glucose and fructose up to 40 weeks. A mouse subgroup was treated with carbon tetrachloride to accelerate fibrosis development. Animals were sacrificed at different time-points to reproduce the full spectrum of histological damage found in human disease, including steatosis (NAFL) and steatohepatitis (NASH) with and without significant fibrosis. Blood and liver tissue samples were obtained from patients (n=195) whose NAFLD severity was assessed histologically. Lipidomic analysis was performed using liquid chromatography-tandem mass spectrometry.ResultsTriglyceride, cholesterol ester and dihydrosphingolipid levels were increased in the liver of model mice in association with the degree of steatosis. Dihydroceramide concentrations increased with the histological severity of the disease in liver samples of mice (0.024 ± 0.003 vs 0.049 ± 0.005, non-NAFLD vs NASH-fibrosis, p<0.0001) and patients (0.105 ± 0.011 vs 0.165 ± 0.021, p=0.0221). Several dihydroceramide and dihydrosphingomyelin species were increased in plasma of NAFLD patients and correlated with accumulation of liver triglycerides.ConclusionsDihydrosphingolipids accumulate in the liver in response to increased free fatty acid overload and are correlated with progressive histological damage in NAFLD. The increase in dihydrosphingolipids is related to upregulation of hepatic expression of enzymes involved inde novosynthesis of ceramides.HIGHLIGHTSNeutral lipids and dihydrosphingolipids accumulate in liver in correlation with the histological severity of NAFLD in both mice and humans.The ceramide pathway is stimulated to alleviate the free fatty acid excess in liver of NAFLD models.Appearance of significant fibrosis is associated with reduced concentrations of neutral lipids but not dihydrosphingolipids in a mouse model of NAFLD.