Accumulation of dihydrosphingolipids and neutral lipids is related to steatosis and fibrosis damage in human and animal models of non-alcoholic fatty liver disease

Background Dihydrosphingolipids 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. Methods Mice 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. Results Triglyceride, 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. Conclusions Dihydrosphingolipids 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 in de novo synthesis of ceramides. HIGHLIGHTS ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by grants from the Instituto de Salud Carlos III, Spain (PI18/01152, PI21/01173 & and PI20/00505), the Ministerio de Ciencia e Innovación, Spain (RTI2018-098113-B-I00, Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020) and CIBER de Fisiopatología de la Obesidad y Nutrición, CIBEROBN, (OBN-18PI032018); all the grants co-financed by the European Development Regional Fund (ERDF, A way to make Europe). CIBER de Fisiopatología de la Obesidad y Nutrición is an initiative of ISCIII. B.B. is supported by pre-doctoral contracts (PEJD-2017-PRE/BMD-4142 and PEJD-2019-PRE/BMD-15962) of the Comunidad Autónoma de Madrid (CAM). R.B. and J.M-B. is a researcher from FIBio-HRC supported by Consejería de Sanidad (CAM). BR-M was supported by the Miguel Servet Type I program (CP19/00098, ISCIII, Spain; co-funded by the Fondo Europeo de Desarrollo Regional-FEDER) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of Hospital Universitario Ramón y Cajal gave ethical approval for this work (ref: EC 276-14 & ref: ES-280790002001) Ethics committee/IRB of Hospital Clínico Universitario Virgen de la Araixaca gave ethical approval for this work (ref: ref: 2020-2-4-HCUVA) Ethics committee/IRB of Hospital Clínico Universitario Virgen de la Victoria de Málaga gave ethical approval for this work (ref: S2200002) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors