乙型肝炎病毒通过自分泌运动因子/溶血磷脂酸信号损伤对糖稳态的作用及其机制研究

Hepatitis B virus impairs glucose homeostasis via autotaxin/lysophosphatidic acid signaling

摘要:

目的 探讨乙型肝炎病毒（HBV）通过自分泌运动因子（ATX）/溶血磷脂酸（LPA）信号对糖稳态的损伤作用及相关机制。方法 利用基因芯片数据库分析HBV对ATX表达的影响。Western blotting检测HBV细胞株HepG2215、稳定表达HBV反式调节蛋白HBx和PreS2的HBx-HepG2和PreS2-HepG2细胞、对照组HepG2细胞的ATX蛋白表达水平。双萤光素酶报告基因检测HBx和PreS2对ATX启动子作用。构建稳定表达HBx和Pre-S2的小鼠分泌胰岛素细胞HBx-NIT、PreS2-NIT，检测两者对胰岛素分泌的影响。利用rAAV8-1.3HBV经尾静脉注射C57BL/6小鼠复制HBV小鼠模型，NC为注射同体积生理盐水的C57BL/6小鼠。小鼠按2 g/kg腹腔注射葡萄糖进行糖耐量试验（GTT）。采用液相色谱-质谱联用法、酶联免疫吸附试验和血糖分析仪分别检测小鼠血清LPA、血清胰岛素和血糖浓度。结果 HepG2215细胞ATX蛋白相对表达量高于HepG2（P <0.05）。PreS2与ATX启动子共转染组萤光素酶活性高于ATX启动子转染组（P <0.05），HBX与ATX启动子共转染组的萤光素酶活性高于ATX启动子转染组（P <0.05）。HBx-HepG2细胞ATX蛋白相对表达量高于HepG2细胞（P <0.05），PreS2-HepG2细胞高于HepG2细胞（P <0.05）。添加不同浓度LPA后NIT细胞胰岛素分泌表达量比较，差异有统计学意义（P <0.05）。1～3 μmol/L LPA相对表达量与胰岛素分泌呈负相关（r =-0.990，P <0.05）。HBx-NIT细胞添加Ki16425前的胰岛素水平低于NIT细胞（P <0.05），PreS2-NIT细胞低于NIT细胞（P <0.05）。NIT、HBx-NIT和PreS2-NIT细胞添加Ki16425后的胰岛素水平较添加前高（P <0.05）。实验组血清LPA相对表达量、平均空腹血糖浓度、注射葡萄糖后的60、120 min平均血糖浓度和血糖浓度曲线下面积（AUC）平均值较对照组高（P <0.05）。实验组注射葡萄糖后15 min血清胰岛素浓度、血清胰岛素水平的AUC值较对照组低（P <0.05）。用HBV小鼠GTT血糖AUC绘制的ROC曲线显示，曲线面积为0.770（95% CI：0.556，0.984），特异性为60.00%（95% CI：0.122，0.738），敏感性为90.00%（95% CI：0.555，0.998）。用HBV小鼠空腹血糖浓度绘制的ROC曲线显示，曲线面积为0.865（95% CI：0.703，1.027），特异性为80.00%（95% CI：0.444，0.975），敏感性为60.00%（95% CI：0.262，0.878）。实验组胰岛β细胞功能指数、胰岛素敏感指数均小于对照组，胰岛素抵抗指数大于对照组（P <0.05）。结论 HBV反式调节蛋白HBx和Pre-S2上调ATX的表达，增强ATX/LPA信号，导致胰岛素分泌抑制，糖耐量异常，糖稳态受损。

Abstract:

Objective To investigate the effects and underlying mechanisms of hepatitis B virus on impairing glucose homeostasis through the autotaxin (ATX) / lysophosphatidic acid (LPA) signaling.Methods Gene expression databases were used to analyze the effect of HBV on the expression of ATX. Western blotting (WB) was performed to detect the protein expressions of ATX in HBV-expressing cell line HepG2215, HBX-HepG2 and PRES2-HepG2 cells stably expressing HBV trans-regulatory proteins HBx and PreS2, and normal control (NC) HepG2 cells. The dual luciferase reporter assay was applied to detect the effects of HBx and PreS2 on the promoter activity of ATX. HBx-NIT and PreS2-NIT, the mice insulin-secreting cells with stable expressions of HBx and pre-S2, were constructed to detect the effects of HBx and pre-S2 on insulin secretion. Mice models of HBV infection were established by injecting C57BL/6 mice with rAAV8-1.3 HBV via tail veins, while C57BL/6 mice injected with the same volume of normal saline were set as NCs. The glucose tolerance test (GTT) was performed by intraperitoneal injection of glucose at a dosage of 2 g/kg body weight of mice. Serum LPA, serum insulin and blood glucose were measured by liquid chromatography-mass spectrometry, ELISA kits and blood glucose analyzer, respectively.Results The protein expression of ATX in HepG2215 cells was higher than that in HepG2 cells (P < 0.05). The luciferase activity in those co-transfected with PreS2 and ATX promoter was higher than that in those transfected with ATX promoter alone (P < 0.05), and that in those co-transfected with HBX and ATX promoter was also higher compared with that in those transfected with ATX promoter alone (P < 0.05). The protein expression of ATX in HBx-HepG2 cells was higher than that in HepG2 cells (P < 0.05), and that in PreS2-HepG2 cells was higher than that in HepG2 cells (P < 0.05). There were differences in insulin secretion among NIT cells treated with different concentrations of LPA (P < 0.05), and the concentration of LPA was negatively correlated with insulin secretion when it was within the range of 1-3 μmol/L (r = -0.990, P < 0.05). The level of insulin in HBx-NIT cells before treatment with Ki16425 was lower than that in NIT cells (P < 0.05), whereas that in PreS2-NIT cells was lower than that in NIT cells (P < 0.05). The levels of insulin in NIT, HBx-NIT and PreS2-NIT cells after treatment with Ki16425 were higher than those before the treatment (P < 0.05). The serum level of LPA, the mean concentration of fasting blood glucose, and mean concentrations of blood glucose and the areas under curves (AUCs) of blood glucose concentrations 60 and 120 min after glucose injection in the experimental group were higher than those in the control group (P < 0.05). The serum concentration of insulin and the AUC thereof 15 min after glucose injection in the experimental group were lower than those in the control group (P < 0.05). The receiver operating characteristic (ROC) curve based on blood glucose concentrations in the GTT of HBV-infected mice showed an AUC of 0.770 (95% CI: 0.556, 0.984), a specificity of 60.00% (95% CI: 0.122, 0.738), and a sensitivity of 90.00% (95% CI: 0.555, 0.998). The ROC curve based on the concentrations of fasting blood glucose in HBV-infected mice showed an AUC of 0.865 (95% CI: 0.703, 1.027), a specificity of 80.00% (95% CI: 0.444, 0.975), and a sensitivity of 60.00% (95% CI: 0.262, 0.878). The homeostasis model assessment-beta cell and the insulin sensitivity index were lower and the insulin resistance index was higher in the experimental group than in the control group (P < 0.05).Conclusions HBV trans-regulatory proteins HBx and PreS2 up-regulate the expression of ATX and enhance the ATX/LPA signaling, leading to inhibition of insulin secretion, abnormal glucose tolerance and impaired glucose homeostasis.