Mutation Of Beclin1 Acetylation Site At K414R Participates In Glycolipids Metabolic Disorders And Diabetes Complications

Part 1 Mutation of the novel acetylation site at K414R of Beclinl is involved in adipocyte differentiation and lipolysisAim:To investigate the impact of mutation at the acetylation site of Beclinl on adipocyte differentiation and lipolysis.Methods:Healthy male C57BL/6 mice were randomly divided into control group and high-fat feeding group.After 3 months of continuous feeding,the weight of mice in high-fat group was≥ 50 g,then the mice were killed,and the subcutaneous and visceral fat were received.The 3T3-L1 preadipocytes were induced to adipocytes.A lentivirus was used to induce a mutation at K414R of Beclin1.The punctate aggregation of lc3-gfp was observed by immunofluorescence;autophagosome was observed under transmission electron microscope;the contents of free fatty acids and glycerol were detected by kit;the expressions of leptin and adiponectin were detected by ELISA;the expression of Beclinl gene was detected by realtime PCR.Adipocyte differentiation was examined using oil red O staining with or without the K414R mutation.Western blot and immunoprecipitation were used to examine genes related to autophagy(Beclinl and LC3),adipocyte differentiation(CEBPa and PPARy),and lipolysis(FAS,HSL,ATGL and perilipin).Results:Immunoprecipitation showed that Ace-Beclinl levels were increased in 3T3L1 adipocytes differentiation,isoproterenol/TNF-α stimulated lipolysis,subcutaneous and visceral adipose tissue of HFD mice.Treatment with nicotinamide and TSA increased the levels of Ace-Beclin1 and LC3.Beclinl acetylation affects the stability of the Beclinl protein.Transmission electron microscopy revealed a significant decrease in autophagosome by the K414R mutation.The expression of the adipocyte differentiation-related proteins PPARγ,ap2,and CEBPα were decreased by the K414R mutation.The K414R mutation decreased the secretion of leptin and increased the secretion of adiponectin by adipocytes.The K414R mutation increases FAS and perilipin and decreases the lipolytic enzymes HSL,p-HSL,and ATGL.Conclusion:Acetylation of Beclinl is observed in mature adipocytes.The new,unreported acetylation locus K414 was identified.Acetylation of Beclinl is involved in the differentiation and lipolysis of adipocytes.Part 2Mutation of Beclin1 acetylation site at K414 alleviates high glucoseinduced podocyte impairment in the early stage of diabetic nephropathy by inhibiting hyperactivated autophagyBackground:Our group recently reported that a mutation of the novel Beclin1 K414R acetylation site impacts the stability of Beclin1 protein,which decreases autophagy in adipocytes and further impedes adipocyte differentiation and lipolysis.This study was to explore whether Beclin1 acetylation plays a role in the early renal injury induced by high glucose and to further investigate the K414R mutation site in podocytes.Methods:Male Sprague-Dawley rats were randomized to con(control)and DN(diabetic nephropathy)groups.The DN group was induced by a single 55 mg/kg intraperitoneal injection of streptozotocin and fed a high-fat and high-sugar diet(the con group received an equal volume of the vehicle and fed a plain diet),after 7 days of induction,blood glucose levels were measured to confirm the onset of diabetes.Then,at weeks 0 and 4,the biochemical index was assayed and renal cortex tissues were harvested.MPC5 podocytes were cultured in vitro.Beclin1(K414R)-pLVX-ZsGreen1-N1(wild-type or mutant)lentiviral plasmids were transfected into podocytes.Western blot or immunoprecipitation was used to test proteins or the acetylation levels respectively,and immunohistochemistry was used to analyze morphological changes of podocytes.Immunofluorescence was used to detect the aggregation of LC3 puncta.Results:The acetylation level of Beclin1 was upregulated with podocyte injury exacerbated in high glucose at 24h and that a mutation at K414R could inhibit hyperactivated autophagy,which ameliorated podocyte impairment.Conclusion:These findings suggest that the acetylation site at K414 is a critical molecule and drug target and that further research into this area is warranted.