Sphingosine Kinase 2 Deficiency Reduces VLDL Secretion And Accompanied With Hepatocyte Lipid Accumulation

Background Parallel to the global increase in metabolic syndrome,diabetes and obesity,a sharp rise is seen in the prevalence of NAFLD.The spectrum of the disease extends from liver steatosis to nonalcoholic steatohepatitis（NASH）;the latter may progress to advanced liver fibrosis,cirrhosis,or hepatocellular carcinoma（HCC）.The hallmark of hepatic steatosis is a net retention of lipids in the liver,and the factors influencing uptake of fatty acids and de novo lipogenesis,fatty acids esterification and oxidation,glucose metabolism balance,especially export of lipids in very low-density lipoproteins（VLDL）may play an important role.Many studies point to the key role of Sph Ks-S1P pathway in metabolic liver diseases.Two isoforms of sphingosine kinases（Sph Ks）,sphingosine kinase 1（Sph K1）and sphingosine kinase 2（Sph K2）,they play similar roles because of their overlapping enzymatic activity in the conversion of ceramide and sphingosine into S1P;whereas in others,Sph K2 exhibits different or even opposite effects due to distinct tissue distribution and subcellular localization.Although the mechanisms underlying Sph K2,a major isotype of sphingosine kinase in the liver,in hepatocellular carcinoma and liver fibrosis have been extensively,the regulatory mechanisms that couple Sph K2 to VLDL secretion are not well understood.Methods First,we will use immunohistochemistry and Western blotting to explore whether lipid accumulation in hepatocytes is related to Sph K2 in non-alcoholic fatty liver patients and fatty liver mice;secondly,using Sph K2 knockout mice,Western blotting,Oil red staining and hematoxylin-eosin staining to further confirm that Sph K2 was critical importance to lipid accumulation;then,proteomics,immunofluorescence,confocal imaging,and co-immunoprecipitation were used to explore the mechanism of TG accumulation after Sph K2 deletion;finally,liquid chromatography tandem mass spectrometry,oil red staining,Western blotting and other techniques were used to detect whether the lack of S1P led to the obstruction of VLDL secretion and the accumulation of TG in hepatocytes.Results We observe that Sph K2 was down-regulated in human and mice fatty liver.Sph K2-deficiency(Sphk2^-/-)mice exhibited hepatocytes lipid accumulation spontaneously and reduced triglycerides（TG）level in peripheral blood,this is due to the disorder of hepatic VLDL secretion in excluding other related pathways.To further investigate how VLDL secretion was regulated by Sph K2,we extracted primary hepatocytes from Sphk2^-/-mice and WT mice for proteomics,Kyoto encyclopedia of genes and genomes（KEGG）analysis revealed that SNAREs interactions in vesicular transport were enriched,significantly.GO analysis showed that the process of membrane fusion was also enriched.We applied immunofluorescence and co-immunoprecipitation technology,the data suggested that Sph K2 deficiency reduced the level of SNAREs complex,and impaired SNAREs interaction in VTVs transport to Golgi process.The exogenous supplementation of S1P significantly reversed the VLDL accumulation and the decreased levels of STX5a,GS28,Sec22b.Conclusion Our identification of the Sph K2-S1P signaling axis uncovered a new regulatory mechanism by modulating SNAREs interaction to maintain hepatic VLDL secretion.Background Sphingosine kinase 2 is mainly distributed in the nucleus,endoplasmic reticulum and mitochondria.The current research is still mostly focus on the S1 P catalyzed by sphingosine kinase 2,which can interact with HDACs in the nucleus to inhibit the deacetylation of histones,thereby promoting gene transcription.The role of other aspects still needs to be further studied.Methods We used oil red staining,western blotting and other experimental techniques to explore the regulation of Sph K2 on SNAREs complex.Results After inhibiting Sph K2 activity,the transcription level of SNAREs complex in hepatocytes remained unchanged,but the protein level decreased.Cycloheximide tracer experiments further confirmed that the stability of the SNAREs complex decreased after Sph K2 activity was inhibited,which promoted the chaperone mediated autophagy degradation pathway.Through testing a variety of autophagy inhibitors,only MHY1485（m TOR activator）can reverse the VLDL secretion disorder and TG accumulation caused by Sph K2 inactivation.After detecting the key proteins of the m TOR pathway,it was found that the m TOR pathway was down-regulated after inhibiting the activity of Sph K2.In vitro experiments confirmed that supplementing exogenous S1 P reversed the down-regulated state of the m TOR pathway in hepatocytes.Conclusion Our mechanistic study of the Sph K2-S1P-m TOR signaling axis reveals a novel function of intracellular S1 P,which regulates autophagy to maintain hepatic VLDL secretion by promoting m TOR phosphorylation.