CerS5 deficiency promotes liver fibrosis development in non-alcoholic fatty liver disease

Abstract

Background: Hepatocyte lipotoxicity mediated by sphingolipids was considered one of important factors in NAFLD development. Knocking out key enzymes for sphingolipids synthesis, such as DES-1, SPHK1 and CerS6, could reduce hepatocyte lipotoxicity and improve NAFLD progression. Previous studies showed that roles of CerS5 and CerS6 in sphingolipids metabolism were similar, but the role of CerS5 was controversial in NAFLD development. This study aimed to clarify the role and mechanism of CerS5 in NAFLD development.

Methods: Hepatocyte conditional CerS5 knockout (CerS5 CKO) and wild type (WT) mice were fed with standard control diet (SC) and choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) and then divided into four groups: CerS5 CKO-SC, CerS5 CKO-CDAHFD, WT-SC and WT-CDAHFD. RT-PCR, IHC and WB were used to analyze the expression of inflammatory, fibrosis and bile acids (BA) metabolism factors. RNA-seq was used to analyze differences of transcriptional levels of liver molecules among the four groups. Metabolomics was used to measured differences of hepatic BAs among the four groups.

Results: Hepatocyte specific knockout of CerS5 did not increase or reduce the severity of 8-weeks CDAHFD induced hepatic steatosis and inflammation, but significantly worsened the progression of liver fibrosis in these mice. At the molecular level, hepatocyte specific knockout of CerS5 did not increase or reduce expression of hepatic inflammatory factors: CD68, F4/80 and MCP-1, but increased expression of hepatic fibrosis factors: α-SMA, COL1α and TGF-β in mice fed with CDAHFD. Transcriptome analysis showed that hepatocyte specific knockout of CerS5 significantly decreased the expression of hepatic cyp27a1, and decreased expression of cyp27a1 was further validated by RT-PCR and WB. Considering that cyp27a1 was a key enzyme in the alternative pathway of BA synthesis, we further found that hepatic BA pools in CerS5 CKO mice were more conducive to the progression of liver fibrosis, which were characterized by elevated hydrophobic 12α-OH BAs and decreased hydrophilic non-12α-OH BAs.

Conclusion: CerS5 played an important role in the progression of NAFLD related fibrosis, and hepatocyte specific knockout of CerS5 accelerated the progression of NAFLD related fibrosis, which was possibly due to the inhibition of BA synthesis alternative pathway by knocking out hepatocyte CerS5.

Keywords: Bile acids; Ceramide synthases; Liver fibrosis; Non-alcoholic fatty liver disease.