VLDL LIPIDOMIC PROFILING REVEALS ALTERED GLUCOSYLCERAMIDE METABOLISM IN NONALCOHOLIC FATTY LIVER DISEASE RELATED LIVER FIBROSIS

Background: The secretion of very low-density lipoprotein (VLDL) is the primary mechanism by which hepatocytes remove lipids from its cellular organelles into the circulation. It is produced entirely by the liver and has a short-half life in the circulation. Hereby, lipid composition in VLDL provides a unique window to assess hepatocellular lipid homeostasis. This study aims to evaluate how VLDL lipid changes with liver fibrosis in nonalcoholic fatty liver disease (NAFLD).
Methods: Fresh frozen plasma from 157 patients with biopsy proven NAFLD were used to prepare VLDL using ultracentrifugation. Lipidomic analysis was performed on lipids extracted from VLDL using Gas Chromatography / Mass Spectrometry (GC/MS). Lipidome values were normalized using total plasma VLDL particle concentration measured by NMR spectroscopy and batch effect was successfully removed using Limma algorithm. The relationship between lipidome and liver fibrosis was analyzed with multivariate regression in LipidR.
Results: Out of the 1507 analyzed lipid species, we identified 8 significant molecules associated with advanced fibrosis (F3-4) as compared to no or early-stage fibrosis (F0-2). Dihexoacylceramide (CerG2) 39:6, sterol (ST) 44:4 and phosphatidic acid (PA) 32:4 were upregulated while cholesterol ester (ChE) 18:0, sphingomyelin (SM) 47:3, triacylglycerol (TG) 18:1/17:1/20:4, ceramide (Cer) 18:1/26:0 and SM 34:2 were downregulated (Figure 1). While the inverse association between ChE, TG and liver fibrosis have been reported previously, the involvement of Cer derivatives were unexpected. Lipid subspecies analyses revealed a decrease in CerG2 precursors, including Ceramides and Sphingomyelin among those with advanced fibrosis. Meanwhile, CerG2, a type of glycosylated sphingolipids, showed a significant increase. Lipid set enrichment analysis was also performed ranked according to log2-fold change metric and showed enrichment of sterol lipids. There were not significant lipid molecules when comparing PNPLA3 (CT/TT vs CC) and TM6SF2 (CT/TT vs CC) genotypes.
Conclusions: Liver fibrosis in NAFLD is associated with an increase in CerG2 and a reciprocal decrease in SM and Cer in VLDLs. As recent studies have found glycosphingolipids accumulate in hepatocellular carcinoma, further investigations are warranted to elucidate the specific mechanisms through which these molecules contribute to fibrogenesis and carcinogenesis in NAFLD.