Study On Degradation Of Plin5 By CMA Participates In Abnormal Lipolysis In NAFLD

?Background?Nonalcoholic fatty liver disease(NAFLD)represents a spectrum of liver abnormalities,characterized by an increase in intrahepatic triglyceride(TG)content that can progress to inflammation and fibrosis.Nonalcoholic steatohepatitis(NASH)has been the significant cause of liver cirrhosis and predicted to become the second leading indication for liver transplantation in the United States.The pathogenesis of NAFLD is not yet clear,insulin resistance has been reckoned as the major mechanism in the development and progression of NAFLD/ NASH.In recent years,a body of evidence supports that autophagy participates in the onset and development of NAFLD and other metabolic diseases.It is reported that inhibition of autophagy increased TGs and LDs,and loss of autophagy decreased TG breakdown in vitro and in vivo.Moreover,a reverse relationship exists between intracellular lipid and autophagic clearance.This interrelationship may trap hepatocytes in a harmful cycle in which decreased autophagy promotes lipid accumulation that then further suppresses autophagic function,thereby additionally increasing lipid retention.Chaperone mediated autophagy(CMA),one type of autophagy,has been reported to play an important role in abnormal hepatic lipid metabolism.It is related to hepatic steatosis while the function of CMA is blocked,because the lipids accumulated in hepatocytes can't be mobilized to other organs to be used.LAMP-2A is the CMA limiting component.The mouse with a conditional knockout for LAMP-2A shows the accumulation of lipid droplets in hepatocyte with reduced perigonadal white adipose tissue and interscapular brown adipose tissue,negative energy balance,and a switch toward carbohydrate metabolism.Deficient of lamp2 a,leading to the dysfunction of CMA,is mainly responsible for the hepatic steatosis.In detail,abnormal CMA could not obliterate perilipins(PLINs),lipid droplet-associated proteins,which hampers the catabolism of lipid droplets by macroautophagy and cytosolic triglyceride lipase.PLINs,classified as PLIN superfamily,include five members(plin1-5).The grouping of these proteins was based on a 100-amino acid region of high sequence homology near their N termini.Their major function is to maintain lipid homeostasis.Each PLIN protein has unique tissue distribution,subcellular location,and lipid-binding properties,indicative of divergent cellular functions.Plin5 is highly expression in oxidative tissues such as the heart,liver,and skeletal muscle,and suggested an apparent paradoxical role in regulating several components of FA metabolism.Increasing reports have confirmed the role of plin5 in lipid metabolism.For example,perilipin 5 is protective in the ischemic heart and protects heart from oxidative burden by sequestering fatty acid from excessive oxidation.Likewise,plin5 involves into lipid and energy metabolism,and exercise training in skeletal muscle.Its function in liver is poorly understood.It is reported that plin5-null mice shows reduced lipid accumulation,increased inflammatory cytokines and serum level of transaminases,active endoplasmic reticulum stress,and elevated lipid oxidation in liver.In reverse,that is to say perilipin 5improves hepatic lipotoxicity by inhibiting lipolysis.Because of the significant function of lamp2 a and plin5 in lipid metabolism respectively,we hypothesize that there is relation between lamp2 a and plin5.In this study,we will try to illuminate that lamp2 a regulates plin5 by CMA in liver lipolysis.?Objectives?1.To identify the correlation between lamp2 a and plin5 in NAFLD.2.To clarify the function of CMA in droplet degradation and the influence in plin5 expression.3.To investigate the mechanisim that CMA degrades plin5.?Methods?1.The expression of lamp2 a and plin5 in liver of NAFLD patients and high fat diet mice is detected by Western-blot and immunohistochemistry.2.Generate lamp2 a knock-out cell line(Hep G2-lamp2a(-))based on Hep G2 cell,and observe the influence of knocking out lamp2 a for the expression of plin5 and decomposes of lipid droplets.3.The expression of plin5 is detected by Western-blot in Hep G2 cells and Hep G2-lamp2a(-)cells when these cells are handled by lysosomal inhibitors(leupeptin,chloroquine)and agonist(6-hexosamine).4.To investigate that lamp2 a regulates plin5 by CMA in liver lipolysis,co-immunoprecipitation and immunofluorescence are used to confirm the interaction between plin5 and hsc70.?Results?1.The results of Western-blot show that the expression of lamp2 a decreases and plin5 increases in liver from NAFLD.In high fat fed mice,both the Western-blot and immunohistochemistry show consistent changes of lamp2 a and plin5 with NAFLD.2.Lamp2 a knock-out cell line(HepG2-lamp2a(-))is generated successfully by craspre gene editing techniques.Western-blot detection shows,the level of plin5 in Hep G2-lamp2a(-)is increased slightly in regular cultivation(DMEM + Serum)(None)compared to Hep G2 cells.When cells are treated with oil acid(OA)for 8hours,the level of plin5 increased significantly both in Hep G2 cells and Hep G2-lamp2a(-).After 8 hours,cells are incubated with regular media(OA>S+)for 16 hours.In this condition,the level of plin5 increased further in both cell lines.While the cells are incubated with serum-deprived medium(OA>S-),the level of plin5 is decreased significantly in Hep G2,but no obvious alteration in Hep G2-lamp2a(-).Bodipy staining suggests that the changes in number and size of lipid droplets in cells are consistent with level of plin5 in above different conditions.Analysis about TG in both cell lines shows similar alterations with lipid droplets and plin5.These results indicate that lamp2 a deficiency leads to the disorders of plin5 degradation and decomposes of lipid droplets in hepatocytes.3.When cells are handled by lysosomal inhibitors,the level of plin5 increased in Hep G2 cells but no obvious alteration in Hep G2-lamp2a(-)cells.When cells are handled by lysosomal agonist,the level of plin5 decreased in Hep G2 cells and still no obvious alteration in Hep G2-lamp2a(-)cells.These results show that the degradation of plin5 is dependent on lysosome.4.Hsc70 is another important element in CMA.Co-immunoprecipitation test suggests that there is interaction between hsc70 and plin5.Immunofluorescence test suggests that there is colocalization between hsc70 and plin5.?Conclusion?This study indicates that the degradation of plin5 is completed by CMA in lysosome.It is possible that the decrease of lamp2 a in liver leads to the degradation disorder of plin5 and other plins,which results in blocked decomposes and accumulation of triglyceride in hepatocytes,contributes to hepatic steatosis.