Liver phosphatidylinosital 3-kinase/protein kinase B pathway is involved in the decrease of insulin sensitivity in rats with fetal growth restriction

目的 探讨肝脏组织磷脂酰肌醇3-激酶/蛋白激酶B(phosphatidylinosital 3-kinase/protein kinase B,PI3-K/AKT)信号通路在胎儿生长受限(fetal growth restriction,FGR)大鼠胰岛素敏感性降低中的作用. 方法 母鼠受孕后第1天始随机分为对照组和低蛋白组,各10只.低蛋白组孕鼠采用低蛋白饮食法(粗蛋白含量为8.00％)建立FGR仔鼠模型.测定对照组和低蛋白组FGR仔鼠生后3、7、14、30、60及90 d(每组每个时间点取雄性仔鼠8只)空腹血浆葡萄糖和血清胰岛素,计算胰岛素抵抗指数及胰岛素敏感指数.实时荧光定量聚合酶链反应技术检测雄性仔鼠生后7、14、30、60及90 d肝脏组织胰岛素受体底物1、2和葡萄糖转运蛋白4 mRNA表达水平,采用Western印迹技术测定胰岛素受体底物1、PI3-K(p110β亚基)、AKT、磷酸化AKT蛋白表达水平.采用简单相关及多重线性回归分析肝脏组织中PI3-K/AKT信号通路关键分子表达改变与胰岛素敏感性变化间的关系. 结果 (1)低蛋白组新生仔鼠平均出生体重为(4.92±0.36)g,低于对照组的(6.43±0.59)g,差异有统计学意义(t=14.73,P＜0.05).低蛋白组仔鼠中FGR发生率为88.2％(97/110),其中雄性仔鼠FGR发生率为94.1％ (48/51).(2)生后60 d时FGR仔鼠空腹血浆葡萄糖开始高于对照组,直至90 d.FGR仔鼠空腹血清胰岛素和胰岛素抵抗指数30 d时显著高于对照组,持续至90 d.FGR仔鼠胰岛素敏感指数自30 d始即显著低于对照组,直至90 d,差异均有统计学意义(P均＜0.05).(3)与对照组相比,FGR仔鼠7d时胰岛素受体底物1、2 mRNA表达均显著降低(0.45±0.02与0.68±0.03,t=16.633,P＜0.05;0.34±0.10与0.70±0.19,t=4.864,P＜0.05),并持续至生后90 d(0.48±0.03与0.59±0.05,t=5.237,P＜0.05;0.49±0.20与0.70±0.11,t=2.253,P＜0.05)；胰岛素受体底物1、PI3-K及磷酸化AKT蛋白表达自14d时出现降低(0.22±0.05与0.52±0.11,t=7.024,P＜0.05;0.46±0.03与0.97±0.08,t=17.508,P＜0.05;0.62±0.10与0.89±0.08,t=6.100,P＜0.05),持续至生后90 d(1.11±0.08与1.32±0.14,t=3.714,P＜0.05;0.63±0.07与1.00±0.19,t=5.206,P＜0.05;0.28±0.03与0.45±0.10,t=4.880,P＜0.05).(4)FGR仔鼠磷酸化AKT蛋白表达量与胰岛素敏感指数呈正相关(r=0.704,P＜0.05)；磷酸化AKT蛋白表达量分别与空腹血浆葡萄糖、空腹血清胰岛素和胰岛素抵抗指数变化呈负相关(r分别为-0.609、-0.561和-0.577,P均＜0.05). 结论 FGR仔鼠肝脏组织中PI3-K/AKT信号通路中某些关键分子表达发生变化,可能参与了胰岛素抵抗的发生.Objective To investigate the effect of liver phosphatidylinosital 3-kinase/protein kinase B (PI3-K/AKT) pathway on the decrease of insulin sensitivity in fetal growth restriction (FGR) rats.Methods Twenty pregnant female rats were randomly divided into two groups one day after conception:normal-protein group and low protein group (n=10,respectively).Rats in low-protein group was given low protein diet (8.00％ protein) during pregnancy to build FGR model,while normal-protein group was given normal protein diet (20.00％ protein).On day 3,7,14,30,60 and 90 after birth,fasting blood samples of 8 male FGR offsprings from low-protein group and 8 normal offsprings from normal-protein group were collected to measure fasting plasma glucose and insulin level.Then insulin resistance index and insulin sensitivity index were calculated to determine insulin sensitivity.On day 7,14,30,60 and 90 after birth,liver tissue of 8 male FGR and normal offsprings were collected,insulin receptor substrate 1,2 (IRS1/IRS2)and glucose transporter 4 (GLUT4) mRNA expression were measured by real-time fluorescence polymerase chain reaction and the protein expressions of IRS1,PI3-K (subunit p110β),and AKT and phosphorylated AKT (pAKT) were measured by Western blot.The relationships between the expression changes of key molecules of PI3-K/AKT pathway and insulin sensitivity were analyzed by correlation and multiple linear regression method.Results (1) Mean birth weight of baby rats in low-protein group was significantly lower than that of normal-protein group [(4.92±0.36) g vs (6.43±0.59) g,t=14.73,P＜0.05].The incidence of FGR in low-protein group was 88.2％ (97/110); and for male offsprings,it was 94.1 ％ (48/51).(2) Compared to normal offsprings,fasting plasma glucose levels of male FGR offsprings were significantly higher from the age of 60 days to 90 days.Insulin levels and insulin resistance index were significantly higher and insulin sensitivity index was lower from the age of 30 days to 90 days,P＜0.05 respectively.(3) Compared to normal offsprings,IRS1 (0.45 ± 0.02 vs 0.68± 0.03,t=16.633,P＜0.05) and IRS2 mRNA (0.34±0.10 vs 0.70±0.19,t=4.864,P＜0.05) expressions in FGR offsprings were lower from day 7 after birth to day 90 (0.48±0.03 vs 0.59±0.05,t=5.237,P＜0.05; 0.49±0.20 vs 0.70±0.11,t=2.253,P＜0.05).There were no differences in expressions of GIUT4 mRNA and AKT protein between two groups (P＞ 0.05).IRS1,PI3-K and pAKT protein expressions of FGR offsprings decreased significantly from day 14 (0.22±0.05 vs 0.52±0.11,t=7.024,P＜0.05; 0.46±0.03 vs 0.97±0.08,t=17.508,P＜0.05; 0.62±0.10 vs 0.89±0.08,t=6.100,P＜0.05) to day 90 (1.11±0.08 vs 1.32±0.14,t=3.714,P＜0.05; 0.63±0.07 vs 1.00±0.19,t=5.206,P＜0.05;0.28±0.03 vs 0.45±0.10,t=4.880,P＜0.05).(4) The pAKT protein expression level of FGR rats was positively correlated with insulin sensitivity index (r=0.704,P＜0.05) ; while negatively correlated to the level of fasting plasma glucose (r=-0.609,P＜0.05),fasting insulin (r=-0.561,P＜0.05) and insulin resistance index (r =0.577,P＜ 0.05).Conclusions The changes of some key molecules' expressions of PI3-K/AKT pathway in liver might be involved in the insulin resistance in FGR rats.国家自然科学基金; 教育部博士点基金中文核心期刊要目总览(PKU)中国科技核心期刊(ISTIC)012743-7491