鸢尾素经PI3K/AKT/mTOR通路调节自噬对2型糖尿病大鼠胰岛β细胞功能的影响

Effect of Irisin on isletβcell function in type 2 diabetic rats by regulating autophagy through PI3K/AKT/mTOR pathway

目的探讨鸢尾素(Irisin)是否通过磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,AKT)/雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)通路增强自噬,改善2型糖尿病(type 2 diabetes mellitus,T2DM)大鼠胰岛β细胞功能,以期为临床治疗T2DM和代谢综合征等慢性代谢性疾病提供新的思路。方法将30只雄性SD大鼠随机分为正常组(NC)、T2DM组和Irisin干预组(T2DM+Irisin),高脂高糖饮食5周加小剂量(35 mg/kg)链脲佐菌素(streptozotocin,STZ)诱导T2DM大鼠模型。腹腔注射Irisin 8周后,检测大鼠空腹血糖(fasting blood glucose,FBG)、空腹胰岛素(fasting insulin,FINS)、甘油三酯(triglyceride,TG)、总胆固醇(total cholesterol,TC)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C)、高密度脂蛋白胆固醇(high-density lipoprotein cholesterol,HDL-C),并进行腹腔注射葡萄糖糖耐量试验(intraperitoneal glucose tolerance test,IPGTT)、腹腔注射胰岛素耐量试验(intraperitoneal insulin tolerance test,IPITT)及高糖钳夹试验,评估各组大鼠胰岛功能及胰岛素抵抗水平,Western blot法检测大鼠胰腺组织中PI3K/AKT/mTOR通路蛋白及自噬相关蛋白的表达水平,免疫组织化学(immunohistochemistry,IHC)法检测大鼠胰腺组织中胰岛素、微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,MAP1LC3)、家蚕隔离体蛋白1(p62)、溶酶体相关膜蛋白-2(lysosomal associated membrane protein-2,LAMP-2)的表达水平。结果大鼠FBG与干预时间存在交互作用(F=11.751,P=0.000),随着干预时间延长,T2DM+Irisin组大鼠FBG逐渐降低,从干预第4周开始,较T2DM组显著下降(F=1008.870,P=0.000);与NC组相比,T2DM组大鼠FINS水平显著降低(P=0.000),经Irisin干预后,较T2DM组显著升高(P=0.000)。与NC组相比,T2DM组大鼠血清中TC、TG、LDL-C浓度显著升高(P均为0.000),HDL-C浓度显著降低(P=0.000),经Irisin干预后,上述指标均得到改善(P分别为0.010、0.000、0.000和0.000)。Western blot结果显示,与NC组相比,T2DM组p62蛋白表达和LC3Ⅱ/LC3Ⅰ值显著增加(P分别为0.008和0.048),LAMP-2蛋白表达显著降低(P=0.000),经Irisin干预后,LC3Ⅱ/LC3Ⅰ值进一步增加(P=0.000),但p62蛋白表达水平显著降低(P=0.047),LAMP-2蛋白表达显著增加(P=0.000),IHC结果与Western blot结果一致。与NC组相比,T2DM组p-PI3K/PI3K、p-AKT/AKT以及p-mTOR/mTOR值均显著降低(P分别为0.006、0.031和0.000),经Irisin干预后,上述指标均进一步降低(P分别为0.033、0.013和0.000)。结论Irisin可通过PI3K/AKT/mTOR信号通路增强自噬,改善胰岛β细胞功能,为治疗T2DM提供了新的思路。

Objective To investigate whether Irisin improves isletβcells function in rats with type 2 diabetes mellitus(T2DM)by enhancing autophagy through the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway,so as to provide new ideas for clinical treatment of chronic metabolic diseases such as T2DM and metabolic syndrome.Methods Thirty SD male rats were randomly divided into normal group(NC group),T2DM group,andIrisininterventiongroup(T2DM+Irisingroup).High-fatandhigh-sugardietfor 5weekspluslow-dose(35mg/kg)streptozotocin(STZ)induced T2DM rat model.After 8 weeks of intraperitoneal injection of Irisin,rats were tested for fasting blood glucose(FBG),fasting insulin(FINS),triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C).The intraperitoneal glucose tolerance test(IPGTT),intraperitoneal insulin tolerance test(IPITT)and hyperglycemic clamp test were performed to assess the islet function and insulin resistance level of rats in each group.The expression levels of PI3K/AKT/mTOR pathway proteins and autophagyrelated proteins in the pancreas were subsequently detected by Western blot.The expression levels of insulin,microtubuleassociated protein 1 light chain 3(MAP1LC3),p62,and lysosomal associated membrane protein-2(LAMP-2)in rat pancreas were detected by immunohistochemistry(IHC).Results There was an interaction between FBG and intervention time in rats(F=11.751,P=0.000),and the FBG gradually decreased in the T2DM+Irisin group with the prolongation of the intervention time.From the 4th week of intervention,the FBG in the T2DM+Irisin group decreased significantly compared with that in the T2DM group(F=1008.870,P=0.000).Compared with NC group,the serum concentrations of TC,TG,and LDL-C concentrations in the T2DM group significantly increased(each P=0.000),while the HDL-C concentrations significantly decreased(P=0.000).After Irisin intervention,the above indexes were all improved(P=0.010,0.000,0.000 and 0.000,respectively).Western blot results showed that compared with NC group,p62 protein expression and LC3Ⅱ/LC3Ⅰincreased significantly(P=0.008 and 0.048,respectively),and LAMP-2 protein expression decreased significantly(P=0.000)in T2DM group.LC3Ⅱ/LC3Ⅰexpression level further increased after Irisin intervention(P=0.000),but p62 protein level significantly decreased(P=0.047)and LAMP-2 protein expression increased significantly(P=0.000),and the IHC results were consistent with the Western blot results.The levels of p-PI3K/PI3K,p-AKT/AKT and p-mTOR/mTOR decreased significantly in the T2DM group compared with NC group(P=0.006,0.031 and 0.000,respectively),and the above indicators further decreased after Irisin intervention(P=0.033,0.013 and 0.000,respectively).Conclusion Irisin can enhance autophagy through PI3K/AKT/mTOR signaling pathway to improve isletβcells function,providing a new idea for the treatment of T2DM.