The Mechanism Of GBA Enhancing Lipid Degradation Through Sphingolipids In Liver Cells

[Background and objective]Accumulating evidence suggests a link between metabolic syndrome such as non-alcoholic fatty liver(NAFLD)disease in the progression of Parkinson’s disease(PD)and other neurodegenerative diseases.PD is a motor neurodegenerative disease,but in the development of PD,there are also many non-motor clinical manifestations.Body weight and Body mass index are generally lower in PD patients compared to healthy individuals,especially in patients with severe PD.BW regulation is closely related to lipid metabolism.Abnormal lipid metabolism will cause disorder of nutrition and energy metabolism,resulting in abnormal increase or decrease of body weight,and seriously disturb human health.PD related genes or related pathways involved have been reported to be related to lipid metabolism.Homozygous mutation of GBA,the encoding gene of Glucosylceramidase beta,leads to Gaucher disease(GD),but both GD and GD carriers(with heterozygous GBA mutations)are important risk factors for the incidence of PD.Studies have shown that total plasma cholesterol levels in blood samples of type 1 GD patients are lower,but LDL cholesterol,HDL cholesterol and triglycerides are significantly higher than those in normal subjects.At the same time,Ceramide,as a key bioactive sphingolipid,is also involved in the regulation of fat metabolism through multiple pathways,and one of the main production pathways of Ceramide is generated by the hydrolysis of upstream metabolic substrates by GBA.Therefore,GBA,a risk gene for PD,is likely to be involved in the regulation of fat metabolism through spingolipids,but its molecular mechanism remains unclear.This study will investigate the relationship between GBA and lipid degradation,and provides a new idea for the prevention and treatment of diseases related to abnormal lipid metabolism.[Methods]We used sodium oleate and sodium palmitate to induce steatosis in human normal liver cells(L02 cells)to construct a cell model of NAFLD Then the changes of lipid droplet(LD)number after GBA function loss were detected by immunofluorescence,and the changes of lipid droplet membrane protein after GBA function loss were detected by western blot.At the same time,GBA was overexpressed in cells to explore the role of GBA in lipid degradation.To investigate the mechanism of GBA enhancing lipid degradation,CRISPR/Cas9 technique was used to construct GBA Knockout(KO)L02 cell lines.[Results]After Knockdown GBA,we found that the accumulation of LD in the cytoplasm was more serious than that of the control group.Through the follow-up experiments of over-expressing GBA and LD degradation,it was found that GBA participated in the degradation process of LD.After repeating the previous immunofluorescent LD accumulation phenotype using GBA KO cell lines,we also exogenously overexpressed Wildtype(WT)GBA to rescue LD accumulation.At the same time,the quantitative detection of kit found the increase of triglyceride in L02 cells after GBA KO,and the increase of lipid droplets membrane protein after GBA KO was found by Western blot,both of which could be rescued by WT GBA.More importantly,we found that exogenously supplemented downstream Ceramide,Sphingosine(Sph),and sphingosine-1-phosphate(S1P)can all rescue or partially rescue GBA KD or KO induced lipid droplet accumulation phenotypes.In the mechanism study,we found that the protein expression level of lysosomal acid lipase in the lipophagy pathway was decreased after GBA KO,and exogenous overexpression of LAL could restore the accumulation of lipid droplets induced by GBA KO.At the same time,we also found in our model that the loss of GBA function leads to the damage of autophagy pathway,and the lysosomal membrane protein(LAMP1)is increased after GBA KO and can be rescue by overexpression of WT GBA.Real-time PCR also detected the decrease of mRNA levels of two sphingolipase enzymes in lysosome of L02 cells after GBA KO,which means the metabolism of sphingolipids in lysosome of L02 cells was disturbed after GBA KO.[Conclusions]GBA is involved in lipid metabolism by enhancing lipid degradation in hepatocytes through sphingolipids,and the mechanism of which may be related to the increase of LAL expression in the lipophagy pathway.