二甲双胍对胰岛素抵抗小鼠肝组织中胎球蛋白A表达及磷脂酰肌醇3-激酶/蛋白激酶B信号通路的影响

Effect of metformin on the expression of fetuin A and phosphatidylinositol 3-kinase/protein kinase B signal pathway in the liver tissue of insulin resistant mice

目的探讨二甲双胍对胰岛素抵抗(IR)小鼠肝组织中胎球蛋白A(FA)表达及磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路的影响。方法将30只无特定病原体级健康雄性小鼠适应性喂养1周后,随机选择10只小鼠为正常组,给予普通饲料喂养8周;另外20只小鼠给予高脂饲料喂养8周,制备IR模型。将造模成功的16只小鼠随机分为模型组(n=8)和二甲双胍组(n=8),二甲双胍组小鼠给予二甲双胍灌胃,102.75 mg·kg^(-1),每日1次,连续给药4周;正常组和模型组小鼠给予同等剂量的生理盐水灌胃。造模8周期间和给药4周期间,每周称量小鼠体质量。给药4周后,3组小鼠禁食12 h,采用腹腔注射葡萄糖耐量实验(IPGTT)检测小鼠糖耐量。给药4周后,使用小鼠胰岛素酶联免疫吸附试验法测定小鼠血清空腹胰岛素(FINS)水平,并计算稳态模型评估-胰岛素抵抗指数(HOMA-IR);采用实时荧光定量聚合酶链式反应法检测小鼠肝组织中胎球蛋白A(FA)、PI3K、Akt mRNA相对表达量;采用Western blot法检测小鼠肝组织中FA、PI3K、磷酸化PI3K(p-PI3K)、Akt、磷酸化Akt(p-Akt)蛋白相对表达量。结果造模8周后,模型组和二甲双胍组小鼠的体质量显著高于正常组(P<0.05);给药1、2、3、4周后,模型组和二甲双胍组小鼠的体质量显著高于正常组(P<0.05);给药3、4周后,二甲双胍组小鼠的体质量显著低于模型组(P<0.05)。给药4周后,模型组和二甲双胍组小鼠的IPGTT曲线下面积显著高于正常组,二甲双胍组小鼠的IPGTT曲线下面积显著低于模型组(P<0.05)。给药4周后,模型组小鼠的FINS水平及HOMA-IR显著高于正常组和二甲双胍组(P<0.05);给药4周后,二甲双胍组与正常组小鼠的FINS水平及HOMA-IR比较差异无统计学意义(P>0.05)。给药4周后,模型组小鼠肝组织中FA、PI3K、Akt mRNA相对表达量及FA蛋白相对表达量显著高于正常组和二甲双胍组,p-PI3K/PI3K、p-Akt/Akt显著低于正常组和二甲双胍组(P<0.05);二甲双胍组与正常组小鼠肝组织中FA、PI3K、Akt mRNA相对表达量、FA蛋白相对表达量及p-PI3K/PI3K、p-Akt/Akt比较差异无统计学意义(P>0.05)。结论二甲双胍可改善IR小鼠的肥胖状态,提高机体对血糖浓度的调节能力,降低FINS水平及HOMA-IR,其机制可能为二甲双胍通过降低肝脏FA表达,激活PI3K/Akt通路,从而缓解IR。

Objective To investigate the effect of metformin on the expression of fetuin A(FA)and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signal pathway in the liver tissue of insulin resistant(IR)mice.Methods Thirty healthy male mice without specific pathogens were adaptively fed for one week,then 10 mice were randomly selected as the normal group,fed with normal diet for 8 weeks,and the other 20 mice were fed with high-fat diet for 8 weeks to prepare the IR model.Sixteen mice with successful IR model were randomly divided into the model group(n=8)and the metformin group(n=8).The mice in the metformin group were given metformin by gavage,102.75 mg·kg^(-1),once a day,for 4 consecutive weeks;the mice in normal group and the model group were given the same dose of normal saline by gavage.During the 8-week modeling period and the 4-week administration period,the body weight of mice was weighed weekly.After 4 weeks of administration,the mice in the three groups were fasted for 12 hours,then the glucose tolerance was measured by intraperitoneal injection glucose tolerance test(IPGTT).After 4 weeks of administration,the level of fasting insulin(FINS)in serum of mice was measured with insulin enzyme-linked immunosorbent assay method,and the homeostasis model assessment-estimated insulin resistance(HOMA-IR)was calculated;the relative expression of fetuin A(FA),PI3K,Akt mRNA in liver tissue of mice was detected by real-time fluorescence quantitative polymerase chain reaction;the relative expression of FA,PI3K,phosphorylated PI3K(p-PI3K),Akt and phosphorylated Akt(p-Akt)in liver tissue of mice was detected by Western blot.Results After 8 weeks of modeling,the body weight of mice in the model group and the metformin group was significantly higher than that in the normal group(P<0.05);after 1,2,3,4 weeks of administration,the body weight of mice in the model group and metformin group was significantly higher than that in the normal group(P<0.05);after 3 and 4 weeks of administration,the body weight of mice in the metformin group was significantly lower than that in model group(P<0.05).After 4 weeks of administration,the area under the curve of IPGTT of mice in the model group and metformin group was significantly higher than that in the normal group(P<0.05),and the area under the curve of IPGTT of mice in the metformin group was significantly lower than that in the model group(P<0.05).After 4 weeks of administration,the FINS level and HOMA-IR of mice in the model group were significantly higher than those in the normal group and metformin group(P<0.05);after 4 weeks of administration,there was no significant difference in FINS level and HOMA-IR between the metformin group and the normal group(P>0.05).After 4 weeks of administration,the relative expression of FA,PI3K,Akt mRNA and FA protein in liver tissues of mice in the model group were significantly higher than those in the normal group and metformin group,while p-PI3K/PI3K and p-Akt/Akt in liver tissues of mice in the model group were significantly lower than those in the normal group and metformin group(P<0.05);there was no significant difference in the relative expression of FA,PI3K,Akt mRNA,FA protein and p-PI3K/PI3K and p-Akt/Akt in liver tissues of mice between the metformin group and the normal group(P>0.05).Conclusion Metformin can improve the obesity status of IR mice,improve the body′s ability to regulate blood glucose concentration,and reduce FINS level and HOMA-IR.The mechanism may be that metformin can alleviate IR by reducing the expression of FA and activating PI3K/Akt pathway in the liver.