内脂素通过PI3K/Akt/FoxO1信号通路对糖尿病KKAy小鼠骨骼肌糖脂代谢及胰岛素抵抗的影响

Effects of Visfatin on glycolipid metabolism and insulin resistance in the skeletal muscle of diabetic KKAy mice via the PI3K/Akt/FoxO1 signaling pathway

目的探讨内脂素对糖尿病KKAy小鼠骨骼肌糖脂代谢及胰岛素抵抗的影响,初步分析其通过PI3K/Akt/FoxO1途径对糖尿病小鼠骨骼肌组织可能的作用机制。方法8周龄雄性C57BL/6J小鼠随机分为普食(ND)组、普食+内脂素(ND+V)组、高脂(HFD)组和高脂+内脂素(HFD+V)组,同周龄雄性KKAy小鼠成模后随机分为糖尿病(DM)组和糖尿病+内脂素(DM+V)组,其中ND+V组、HFD+V组和DM+V组连续3 d腹腔注射内脂素重组蛋白。检测各组小鼠空腹血糖(FBG)、餐后血糖(PBG)、总胆固醇(TC)、甘油三酯(TG)、游离脂肪酸(FFA)及空腹胰岛素(FIns),并进行葡萄糖耐量实验(GTT)、胰岛素耐量实验(ITT),计算曲线下面积(AUC)及稳态模型评估的胰岛素抵抗指数(HOMA-IR);RT-qPCR及Western blot检测磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(Akt)、叉头框转录因子1(FoxO1)、丙酮酸脱氢酶激酶4(PDK4)和磷酸烯醇丙酮酸羧激酶(PEPCK)等相关分子的表达水平。结果分别与ND组和HFD组比较,DM组小鼠的体重、摄食、FBG、PBG、TC、TG、FFA、FIns、AUC_(GTT)、AUC_(ITT)及HOMA-IR水平均显著增高(P<0.05)。与DM组相比,DM+V组FBG、TC、TG、AUC_(GTT)、AUC_(ITT)及HOMA-IR水平均显著降低(P<0.05)。分别与ND组和HFD组比较,DM组PI3K、Akt的mRNA表达水平均显著降低(P<0.001),而FoxO1的mRNA表达水平显著增高(P<0.05);与DM组比较,DM+V组PI3K及Akt的mRNA表达均显著增高,FoxO1的mRNA表达显著降低(P<0.01)。与ND组比较,DM组PI3K_(110α)、p-Akt蛋白表达水平显著降低,FoxO1、p-FoxO1和PDK4的蛋白表达水平均显著增高(P<0.05);与HFD组相比,DM组的p-Akt、p-FoxO1的蛋白表达水平均显著降低,FoxO1、PDK4和PEPCK的蛋白表达水平均显著增高(P<0.05);与DM组比较,DM+V组中PI3K_(110α)、p-Akt及p-FoxO1的蛋白表达均显著增高,而FoxO1、PDK4和PEPCK的蛋白表达水平均显著降低(P<0.05)。结论内脂素可能通过PI3K/Akt/FoxO1途径下调糖尿病小鼠骨骼肌PDK4的表达,发挥改善糖脂代谢和胰岛素抵抗的作用。

Objective To investigate the effects of Visfatin on glycolipid metabolism and insulin resistance in the skeletal muscle of diabetic KKAy mice,preliminarily analyze the possible mechanism of action through the PI3 K/Akt/FoxO1 pathway.Methods Eight-week-old male C57BL/6 J mice were randomly divided into a normal diet(ND)group,a normal diet+Visfatin(ND+V)group,a high-fat(HFD)group,and a high-fat+Visfatin(HFD+V)group.When the model was generated,male KKAy mice of the same age were randomly divided into a diabetes mellitus(DM)group and diabetes mellitus+Visfatin(DM+V)group.Among them,the ND+V,HFD+V and DM+V groups were intraperitoneally injected with recombinant Visfatin protein for 3 consecutive days.Fasting blood glucose(FBG),postprandial blood glucose(PBG),total cholesterol(TC),triglyceride(TG),free fatty acids(FFA)and fasting insulin(FIns)were detected in each group of mice.The glucose tolerance test(GTT)and insulin tolerance test(ITT)were measured;meanwhile,the area under curve(AUC)and homeostasis model assessment for insulin resistance(HOMA-IR)were calculated.At the same time,the expression levels of phosphatidylinositol 3 kinase(PI3K),protein kinase B(Akt),forkhead box transcription factor 1(FoxO1),pyruvate dehydrogenase kinase 4(PDK4),phosphoenolpyruvate carboxykinase(PEPCK)and other related molecules were detected by RT-qPCR and Western blot.Results Compared with the ND group and the HFD group,the body weight,food intake,FBG,PBG,TC,TG,FFA,FIns,AUCGTT,AUCITT and HOMA-IR levels in the DM group were significantly increased(P<0.05).Compared with DM group,FBG,TC,TG,AUCGTT,AUCITT,and HOMA-IR levels in DM+V group were significantly reduced(P<0.05).Compared with the ND group and the HFD group,the mRNA expression levels of P13 K and Akt in the DM group were significantly decreased(P<0.001),but the mRNA expression levels of FoxO1 were increased(P<0.05).Compared with the DM group,P13 K and Akt mRNA expression in the DM+V group was obviously increased,but the mRNA expression of FoxO1 was significantly reduced(P<0.01).Compared with the ND group,PI3K110αand p-Akt expression in the DM group was reduced,and the protein expression levels of FoxO1,p-FoxO1 and PDK4 were increased.Compared with the HFD group,p-Akt and p-FoxO1 expression was lower in the DM group,and the protein expression levels of FoxO1,PDK4 and PEPCK were increased(P<0.05).While compared with the DM group,PI3K110α,p-Akt and p-FoxO1 expression were increased in the DM+V group,but the protein expression levels of FoxO1,PDK4 and PEPCK were reduced(P<0.05).Conclusions Visfatin may downregulate PDK4 expression in the skeletal muscle of diabetic mice through the PI3 K/Akt/FoxO1 pathway and has a role in improving glucolipid metabolism and insulin resistance.