PPARδ调控自噬在GLP-1受体激动剂艾塞那肽改善胰岛β细胞功能中的作用  

Role of PPARδ in improving β-cell function by GLP-1 receptor agonist exenatide treatment via regulating autophagy

作　　者：苏燕娜 林倍思[1] 陈亚兰 刘子瑜 甘露 徐芬[1] 许雯[1] SU Yan-na;LIN Bei-si;CHEN Ya-lan;LIU Zi-yu;GAN Lu;XU Fen;XU Wen(Department of Endocrine and Metabolic Diseases,The Third Affiliated Hospital of Sun Yat-sen University,Guangdong Key Laboratory of Diabetes Prevention and Treatment,Guangzhou 510630,China)

机构地区：[1]中山大学附属第三医院内分泌与代谢病学科,广东省糖尿病防治重点实验室,广东广州510630

出　　处：《中国病理生理杂志》2022年第10期1737-1746,共10页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81770821);广州市科技计划项目(No.202102010175)。

摘　　要：目的:探讨胰高血糖素样肽1(glucagon-like peptide-1,GLP-1)受体激动剂艾塞那肽(exenatide/exendin-4,Exe)对胰岛β细胞功能的作用及机制。方法:通过高脂饮食(high-fat diet,HFD)喂养C57BL/6J小鼠12周构建2型糖尿病(type 2 diabetes mellitus,T2DM)模型后随机分为HFD+生理盐水(saline)组和HFD+Exe(24 nmol·kg-1·d-1)组,另设正常饮食对照(normal diet control,NC)组,每组10只。监测体重和空腹血糖(fasting plasma glucose,FPG)。HFD喂养12周及Exe干预8周时行腹腔注射葡萄糖耐量实验(intraperitoneal glucose tolerance test,IPGTT)及腹腔注射胰岛素耐量实验(intraperitoneal insulin tolerance test,IPITT)。实验结束时留取胰腺组织行微管相关蛋白1轻链3B(microtubule-associated protein 1 light chain 3B,LC3B)免疫荧光染色,提取各组小鼠原代胰岛总蛋白检测过氧化物酶体增殖物激活受体δ(peroxisome proliferator-activated receptorδ,PPARδ)、LC3B-II和P62的蛋白水平。采用棕榈酸(palmitic acid,PA)诱导小鼠胰岛素瘤NIT-1细胞,分别予Exe(20 nmol/L)和GW501516(10μmol/L)干预24 h后行葡萄糖刺激胰岛素分泌(glucose-stimulated insulin secretion,GSIS)实验,并检测PPARδ、LC3B-II和P62蛋白水平。另外,取部分NIT-1细胞经PA诱导后分别予Exe或/和GSK0660(1μmol/L)干预24 h,并且进一步通过CRISPR/Cas9基因编辑技术构建PPARδ基因敲除(PPARδ-KO)细胞,经PA诱导后予Exe干预24 h,验证PPARδ在Exe激活β细胞自噬中的作用。结果:在HFD喂养12周后,小鼠体重和FPG显著升高,糖耐量受损明显,成功构建小鼠T2DM模型;与HFD+saline组相比,Exe治疗可明显降低T2DM小鼠体重和FPG,改善受损糖耐量(P<0.05)。与NC组相比,HFD+saline组胰岛LC3B荧光表达降低,胰岛LC3B-II和PPARδ的蛋白表达量降低,P62蛋白表达升高;与HFD+saline组相比,Exe治疗后胰岛LC3B荧光表达增加,LC3B-II和PPARδ蛋白表达量升高,P62蛋白表达量降低(P<0.05)。此外,与PA组相比,Exe和GW501516干预组在高糖刺AIM:To investigate the effect of glucagon-like peptide-1(GLP-1)receptor agonist exenatide/exendin-4(Exe)onβ-cell function and the underlying mechanisms.METHODS:Type 2 diabetes mellitus(T2DM)C57BL/6J mice were randomly divided into high-fat diet(HFD)+saline group and HFD+Exe(24 nmol·kg-1·d-1)group after fed with HFD for 12 weeks,and another C57BL/6J mice fed with normal diet served as normal diet control(NC)group,with 10 mice in each group.Body weight and fasting plasma glucose(FPG)were recorded.Intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT)were performed at 12 weeks of HFD and after 8weeks of Exe intervention.At the end of the experiment,the pancreatic tissues were collected for microtubule-associated protein 1 light chain 3B(LC3B)immunofluorescence staining,and the mouse islet were isolated and extracted to detect the protein levels of peroxisome proliferator-activated receptorδ(PPARδ),LC3B-II and P62.The NIT-1 cells after induced by 400μmol/L palmitate acid(PA)were treated with Exe(20 nmol/L)or GW501516(10μmol/L)for 24 h,the glucose-stimulated insulin secretion(GSIS)assay was performed,and the protein levels of PPARδ,LC3B-II and P62were detected.In addition,after induced by PA,mouse insulinoma NIT-1 cells were treated with Exe,GSK0660(1μmol/L)or Exe+GSK0660 for 24 h.Furthermore,in order to gain further evidence for the role of PPARδin promoting autophagy ofβ-cell by GLP-1 receptor agonist,PPARδgene knockout(PPARδ-KO)NIT-1 cells generated by CRISPR/Cas9 technique were treated with Exe for 24 h after PA induction.RESULTS:After 12 weeks of HFD,the body weight and FPG of the mice were significantly increased(P<0.05),and the glucose tolerance was significantly impaired,which suggested that the T2DM model in mice were successfully established.Compared with HFD+saline group,Exe significantly reduced the body weight and FPG,and improved the glucose tolerance.The fluorescence intensity of LC3B in HFD+saline group was decreased compared with NC group.The protei

关 键 词：艾塞那肽 2型糖尿病 过氧化物酶体增殖物激活受体Δ 自噬 胰岛Β细胞 

分 类 号：R587.1[医药卫生—内分泌] R363.1[医药卫生—内科学]