Rab32 Controls Intracellular Lipid Accumulation Through Lipolysis Via Enhancing ATGL Expression In Hepatocytes

Non-alcoholic fatty liver disease(NAFLD)is a kind of disease caused by abnormal lipid accumulation in hepatocytes,which excludes the influence of alcohol abusing and hepatic injury.To date,the pathogenesis of NAFLD remains unclear.The “two hit” hypothesis is the most widely accepted hypothesis until now.The first hit is the abnormal lipid accumulation in hepatocytes caused by insulin resistance and so on.The second hit is caused by the stimulation of intracellular abnormal lipid accumulation,which can lead to NASH,hepatic fibrosis and even HCC.Therefore,to study the molecular mechanism involved in the pathogenesis of NAFLD is quite significant for preventing NAFLD developing to NASH,hepatic fibrosis and even HCC.Rab proteins are the largest subfamily of small GTP binding protein.Rabs involve in kinds of membrane trafficking in intracellular.Rab proteins work through the molecular switch mechanism in vesicular trafficking,participate in the process of vesicular formation,vesicular trafficking,adhesion,anchoring,fusion in cells,which control intracellular material transport and membrane trafficking.Because there are intensive membrane activities in the intracellular abnormal lipid accumulation,it is reasonable to hypothesize that Rabs involve in the process of lipid accumulation and play key roles in regulating intracellular lipids level.To date,several studies have reported that Rabs participate in regulating intracellular lipid accumulation.Rab32 is the only Rabs which can be co-localized with mitochondria,which reveal the possibility of its involving in regulating intracellular energy metabolism.It has been reported that Rab32 involved in the process of regulating lipid accumulation in the fat body and in larval adipose tissue in Drosophila at the condition of starvation.To date,somehow,the possible molecular mechanism of Rab32 regulating lipid accumulation at normal and high-fat condition remains unknown.Additionally,the function of Rab32 in human may be quite different from that in Drosophila.In this case,it is meaningful to study the possible mechanism of Rab32 in regulating lipid accumulation in hepatocytes,which lead us to understand the pathogenesis of NAFLD better.Objection:To study the impacts of Rab32 on intracellular lipid accumulation and its possible regulating mechanismMethod:1.Utilizing eXact-6x His purification system to identifying and analyzing the co-effectors of Rab32In previous study,we constructed eXact-6xHis purification system and identified that it is working in eukaryotic cells.After optimizing the purification protocols,the purifying efficiency has been intensively improved.Hepa1-6plentisEHEGFPRab32 has been constructed.After purification,the purifying effects were identified by SDS-PAGE electrophoresis followed by silver staining.The result suggested that the protein purified can be sent to do a mass spectrum analysis.We sent the sample to do a mass spectrum analysis and found several co-effectors of Rab32.2.Mechanism study of Rab32 in regulating intracellular lipid accumulation in hepatocytesAfter identifying the co-effectors of Rab32 with Mass spectrum analysis,we thought that Rab32 may involve in energy metabolism in cells.Analyzing the expression level of Rab32 from 13 NAFLD patients and 13 normal people in GEO database(GSE48452),we collected liver tissues from 6 NAFLD patient and 6 normal people and analyzed Rab32 expression level with QPCR analysis.Next,we constructed NAFLD cell model with fatty acids(OA: PA=2:1)and detected Rab32 protein level.Results showed that Rab32 was decreased in hepatocytes under high –fat condition.These data suggested that Rab32 expression level changed in lipid accumulation,which reveal that Rab32 may play roles in regulating lipid accumulation.Then,we overexpressed and knockdown Rab32 in hepatocytes,and proved that Rab32 impacts on intracellular lipid accumulation by lipolysis via control the expression of ATGL.Co-IP showed that Rab32 impact on ATGL expression indirectly,which support our mass spectrum analysis result.Conclusion:1.Purifying Rab32 with e Xact-6x His purification system,we identify co-effectors of Rab32 with lable free mass spectrum analysis containing five times sample injection and Decoy analysis to detect the co-effectors.Rab38,PHB1 and PHB2 has been proved to be the possible co-effectors of Rab32,which reveal that Rab32 may participate in intracellular energy metabolism.2.Rab32 expression level analysis from GEO database and QPCR analysis in samples showed that Rab32 is decreased in liver from NAFLD patient.NAFLD cell model was constructed with Hep G2 and L02 cells.Western Blots showed that Rab32 protein level was decreased under high-fat condition.Overexpressing and Knocking down Rab32 showed that only Rab32 ablation decreased intracellular lipid accumulation,while no effect was observed when Rab32 was overexpressed.Next,we proved that Rab32 control intracellular lipid accumulation via lipolysis through affecting ATGL expression.In summary,we concluded that Rab32 control intracellular lipid level through lipolysis via affecting ATGL expression in hepatocytes.