IGF2BP2调控PPAR-γ/GLUT4通路在亚砷酸钠暴露所致HepG2细胞胰岛素抵抗中的作用机制

Mechanism of IGF2BP2 regulation of PPAR-γ/GLUT4 pathway in insulin resistance induced by sodium arsenite exposure in HepG2 cells

  • 摘要:
    背景 砷是一种环境有害物质,会导致肝胰岛素抵抗和肝损伤,增加患2型糖尿病的风险。
    目的 探讨胰岛素样生长因子2 mRNA结合蛋白2(IGF2BP2)是否通过过氧化物酶体增殖物激活受体γ(PPAR-γ)/葡萄糖转运蛋白4(GLUT4)通路参与砷暴露所致的HepG2细胞胰岛素抵抗。
    方法 采用细胞计数试剂盒8(CCK8)检测细胞活力,并确定合适的NaAsO2染毒剂量。用不同浓度的NaAsO2溶液分别处理HepG2细胞24 h以建立细胞砷暴露模型(实验分为四组:0、2、4和8 μmol·L−1组);用pcDNA3.1-IGF2BP2、pcDNA3.1-NC分别处理HepG2细胞6 h后,再使用8 μmol·L−1 NaAsO2处理HepG2细胞24 h以建立IGF2BP2过表达细胞模型(实验分为4组:control组、NaAsO2组、NaAsO2+pcDNA3.1-IGF2BP2组、NaAsO2+pcDNA3.1-NC组);最后使用100 nmol·L−1的胰岛素刺激30 min。采用糖原和葡萄糖含量检测试剂盒测定HepG2细胞内糖原和葡萄糖含量;采用实时荧光定量PCR检测细胞内IGF2BP2的mRNA表达水平;采用免疫印记法(WB)检测HepG2细胞内IGF2BP2、PPAR-γ、GLUT4的蛋白表达水平;采用免疫共沉淀(CO-IP)实验验证IGF2BP2与PPAR-γ以及PPAR-γ与GLUT4的结合情况。
    结果 CCK8实验结果显示NaAsO2浓度与细胞活力存在剂量效应关系,当NaAsO2浓度≥4 μmol·L−1时,较对照组降低(P<0.05);随着NaAsO2染毒剂量的升高,HepG2细胞中葡萄糖消耗量和糖原水平减少,2、4、8 μmol·L−1 NaAsO2处理组与对照组之间的差异有统计学意义(P<0.05);4、8 μmol·L−1 NaAsO2处理组HepG2细胞中IGF2BP2的mRNA表达水平与对照组之间的差异有统计学意义(P<0.05)。IGF2BP2过表达细胞模型中,与对照组相比,NaAsO2组葡萄糖消耗量和糖原水平减少(P<0.05),IGF2BP2的mRNA表达水平以及IGF2BP2、PPAR-γ、细胞膜中GLUT4的蛋白表达水平均下降(P<0.05);与NaAsO2组相比,NaAsO2+pcDNA3.1-IGF2BP2组葡萄糖消耗量和糖原水平增加(P<0.05),并且IGF2BP2的mRNA表达水平以及IGF2BP2、PPAR-γ、细胞膜中GLUT4的蛋白表达水平均上升(P<0.05);CO-IP实验显示IGF2BP2与PPAR-γ以及PPAR-γ与GLUT4蛋白相互作用。
    结论 IGF2BP2通过作用于PPAR-γ/GLUT4通路参与砷暴露所致的HepG2细胞胰岛素抵抗。

     

    Abstract:
    Background Arsenic is an environmentally harmful substance that causes hepatic insulin resistance and liver damage, increasing the risk of type 2 diabetes mellitus.
    Objective To explore whether the insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) is involved in insulin resistance in HepG2 cells after arsenic exposure through the peroxisome-proliferator-activated receptor γ (PPAR-γ) / glucose transporter 4 (GLUT4) pathway.
    Methods Cell viability was determined using cell counting kit 8 (CCK8) and an appropriate NaAsO2 infection dose was determined. A cellular arsenic exposure model of HepG2 cells was established by four concentrations of NaAsO2 solution for 24 h (the experiment was divided into four groups: 0, 2, 4, and 8 μmol·L−1); HepG2 cells were firstly treated with pcDNA3.1-IGF2BP2 and pcDNA3.1-NC respectively for 6 h, then with 8 μmol·L−1 NaAsO2 for 24 h to establish a IGF2BP2 overexpression cell model (the experiment was divided into 4 groups: control, NaAsO2, NaAsO2+pcDNA3.1-IGF2BP2, and NaAsO2+pcDNA3.1-NC); finally the cells were subject to 100 nmol·L−1 insulin stimulation for 30 min. Glycogen and glucose in HepG2 cells were determined by glycogen and glucose assay kits; mRNA expression levels of IGF2BP2 were measured by quantitative real-time PCR; protein expression levels of IGF2BP2, PPAR-γ, and GLUT4 in HepG2 were detected by Western blot (WB); and the binding of IGF2BP2 to PPAR-γ and PPAR-γ to GLUT4 was verified by co-immunoprecipitation (CO-IP) experiment.
    Results The results of CCK8 experiment showed a dose-effect relationship between NaAsO2 concentration and cell viability. When the concentration of NaAsO2 was ≥4 μmol·L−1 , the cell viabilities were lower than that of the control group (P <0.05). With the increasing dose of NaAsO2 infection, reduced glucose consumption and glycogen levels in HepG2 cells were found in the 2, 4, and 8 μmol·L−1 NaAsO2 treatment groups compared to the control group (P <0.05). The difference between the mRNA expression level of IGF2BP2 in the HepG2 cells treated with 4 or 8 μmol L−1 NaAsO2 and the control group was significant (P <0.05). In the IGF2BP2 overexpression cell model, compared with the control group, glucose consumption and glycogen levels were lowered in the NaAsO2 group (P <0.05), the mRNA expression level of IGF2BP2 and the protein expression levels of IGF2BP2, PPAR-γ, and GLUT4 in the cell membrane were all decreased (P <0.05). Compared with the NaAsO2 group, the glucose consumption and glycogen levels were increased in the NaAsO2+pcDNA3.1-IGF2BP2 group (P <0.05), and the mRNA expression level of IGF2BP2 and the protein expression levels of IGF2BP2, PPAR-γ, and GLUT4 in the cell membrane were all increased (P <0.05). The results of CO-IP experiments showed that IGF2BP2 interacted with PPAR-γ as well as PPAR-γ with GLUT4 protein.
    Conclusion IGF2BP2 is involved in arsenic exposure-induced insulin resistance in HepG2 cells by acting on the PPAR-γ/GLUT4 pathway.

     

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