自噬核心蛋白Atg101对脂肪细胞衰老的影响  

Effect of autophagic core protein Atg101 on adipocyte senescence

作　　者：叶雅芬 徐蓉蓉 马静远 杨颖[1] 韩峻峰[1] Ye Yafen;Xu Rongrong;Ma Jingyuan;Yang Ying;Han Junfeng(Department of Endocrinology and Metabolism,Shanghai Sixth People′s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,Shanghai Diabetes Institute,Shanghai 200233,China)

机构地区：[1]上海交通大学医学院附属第六人民医院内分泌代谢科,上海市糖尿病研究所,200233

出　　处：《中华内分泌代谢杂志》2023年第1期48-54,共7页Chinese Journal of Endocrinology and Metabolism

摘　　要：目的初步探究自噬核心蛋白Atg101对白色脂肪细胞衰老的影响。方法构建Atg101敲减的3T3-L1成熟脂肪细胞模型,验证Atg101对自噬相关蛋白LC3和p62蛋白的影响。构建并分析人类皮下脂肪组织的RNA-seq数据库,基于Atg101与其他基因FPKM值的Pearson相关系数(R^(2)>0.4,P<0.050)预测共表达基因集并进行KEGG和Reactome富集分析。构建年轻小鼠(8周龄)和老龄小鼠(18个月龄)模型,实时定量PCR(RT-qPCR)和Western印迹法检测Atg101在腹股沟皮下脂肪和内脏脂肪中的表达水平。进一步通过RNA-seq、Western印迹和RT-qPCR检测白色脂肪细胞衰老相关分泌表型(senescence-associated secretory phenotype,SASP)、细胞周期及线粒体稳态相关基因的表达差异,分析Atg101敲减对脂肪细胞衰老的影响。结果在3T3-L1脂肪细胞敲减Atg101后自噬相关蛋白LC3-Ⅱ显著降低,p62蛋白明显上调,提示细胞自噬受损。KEGG富集分析发现Atg101共表达基因集主要富集于自噬和衰老相关通路;Reactome富集分析发现该基因集与多个细胞周期信号通路相关。RT-qPCR和Western印迹证实Atg101在老龄小鼠皮下脂肪组织中mRNA和蛋白表达水平均下调,内脏脂肪组织蛋白水平也显著降低。最后,白色脂肪细胞模型中Atg101敲减后SASP相关基因表达上调,细胞周期特异性基因表达受限并且细胞周期素依赖性蛋白激酶抑制因子p16、p21表达显著增高,线粒体稳态调节基因表达也受到抑制。结论Atg101可能通过影响自噬活动调控白色脂肪细胞衰老,从而破坏线粒体稳态。Objective To investigate the effect of autophagy related gene Atg101 on white adipocyte senescence.Methods An Atg101 knockdown model of 3T3-L1 mature adipocytes was constructed to probe the effect of Atg101 on autophagy-related proteins LC3 and p62 protein.The RNA-seq database of human subcutaneous adipose tissue was constructed and analyzed,and the co-expressed gene set was predicted based on the pearson correlation coefficient(R^(2)>0.4,P<0.05)between FPKM values of Atg101 and other gene,followed by KEGG and Reactome enrichment analysis.Young mouse(8 weeks old)and old mouse(18 months old)models were established,and the expression levels of Atg101 in inguinal white adipose tissue and epididymal white adipose tissue were detected by quantitative real-time PCR(RT-qPCR)and Western blot.Furthermore,the differences in white adipocyte senescence-associated secretory phenotype(SASP),cell cycle and mitochondrial homeostasis-related genes were detected by RNA-seq,Western blot,and RT-qPCR to analyze the effects of Atg101 silencing on adipocyte senescence.Results The autophagy-related protein LC3-Ⅱexpression was significantly decreased and p62 protein was induced after Atg101 was knockdowned in 3T3-L1 adipocytes,suggesting impaired cell autophagy.KEGG enrichment analysis revealed that Atg101 co-expressed gene set was mainly enriched in autophagy and senescence-related pathways;Reactome enrichment analysis revealed that this gene set was associated with multiple cell cycle signaling pathways.RT-qPCR and Western blot confirmed that both mRNA and protein levels of Atg101 were down-regulated in inguinal white adipose tissue of aging mice,and protein levels in epididymal white adipose tissue were also significantly reduced.Finally,it was further confirmed that SASP-related genes were induced after Atg101 knockdown in white adipocytes,and cell cycle-specific gene expression was restricted and cytokine-dependent protein kinase inhibitors p16 and p21 expressions were significantly increased,while mitochondrial homeostasis regula

关 键 词：Atg101 自噬 白色脂肪细胞 衰老 线粒体稳态 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]