砷对秀丽隐杆线虫糖代谢的影响

Effects of arsenic exposure on glucose metabolism in Caenorhabditis elegans

目的观察慢性砷暴露对秀丽隐杆线虫糖代谢的影响,并探讨其作用机制。方法将同步化后的线虫分别暴露于含0(对照)、5、50μmol/L亚砷酸钠的M9缓冲液中72 h。采用高效液相色谱-串联质谱(UPLC-MS/MS)法进行代谢组学检测,运用偏最小二乘判别分析(PLS-DA)分析比较砷暴露组和对照组线虫代谢产物的差异,并筛选出有意义的差异代谢物,利用京都基因与基因组百科全书(KEGG)查找其影响的相关代谢通路。测定线虫体内葡萄糖水平及糖酵解、糖原合成和分解、糖异生途径相关基因的表达情况。结果PLS-DA分析显示,暴露组与对照组线虫代谢特征存在差异,砷暴露组中葡萄糖-6-磷酸表达量降低,差异具有统计学意义(P<0.05)。50μmol/L亚砷酸钠暴露组线虫体内葡萄糖的含量高于对照组,差异有统计学意义(P<0.05);且随着亚砷酸钠暴露浓度的升高,线虫体内葡萄糖水平呈上升趋势。与对照组相比,各浓度亚砷酸钠暴露组线虫体内糖酵解途径相关基因己糖激酶(hxk-2)、6-磷酸果糖激酶(pfk-1.1)和丙酮酸激酶(pyk-1)mRNA的表达水平均降低,差异有统计学意义(P<0.05);且随着亚砷酸钠暴露浓度的升高,线虫体内hxk-2、pfk-1.1mRNA的表达水平均呈下降趋势。与对照组相比,各浓度亚砷酸钠暴露组线虫体内糖原合成相关基因糖原合酶(gsy-1)mRNA的表达水平均降低,而糖原分解相关基因糖原磷酸化酶(pygl-1)mRNA的表达水平均升高,差异均有统计学意义(P<0.05);且随着亚砷酸钠暴露浓度的升高,线虫体内gsy-1 mRNA的表达水平呈下降趋势,而pyg1-1 mRNA的表达水平呈上升趋势。与对照组相比,各浓度亚砷酸钠暴露组线虫体内糖异生途径相关基因磷酸烯醇式丙酮酸激酶(pck-1)mRNA表达水平降低,而50μmol/L亚砷酸钠暴露组线虫体内果糖二磷酸酶-1(fbp-1)mRNA的表达水平升高,差异均有统计学意义(P<0.05);且随着亚砷酸钠暴露浓度的升高,线虫体内pck-1 mRNA的表达水平均呈下降趋势。结论慢性砷暴露可通过影响糖代谢相关酶的表达导致线虫糖代谢紊乱。

Objective To understand the effects of chronic arsenic exposure on glucose metabolism of Caenorhabditis elegans(C.elegans)and the mechanism.Methods The synchronized C.elegans were randomly assigned into the exposure groups and control group and exposed to sodium arsenate(5μmol/L and 50μmol/L)and M9 buffer for 72 h,respectively.After exposure,the UPLC-MS/MS was used to determine the metabolites.Partial least squares discriminant analysis(PLS-DA)was used to analyze and compare the differences in metabolites between the arsenic-exposed group and the control group,screening out the meaningful differential metabolites,and the KEGG database was used to find relevant metabolic pathways affected by them.Glucose oxidase method was used to determine the content of glucose in nematode tissues.The expression of glycolysis,glycogen synthesis and decomposition,and gluconeogenesis pathway related genes in C.elegans was detected by qRT-PCR.Results PLS-DA analysis showed a significant difference between the metabolic characteristics of the exposure group and the control group.Glucose-6-phosphoric acid content in the exposure group was significantly lower than that in the control group(P<0.05).The glucose content in 50μmol/L arsenite exposure group was significantly higher than that of the control group(P<0.05),and presented a dose dependent manner.Compared with the control group,the expression of glycolysis genes hexokinase(hxk-2),6-phosphate fructokinase(pfk-1.1)and pyruvate kinase(pyk-1)mRNA decreased in arsenic exposure group(P<0.05)with a dose dependent manner.Compared with the control group,the expression of glycogenesis gene glycogen synthase(gsy-1)mRNA decreased in arsenite exposure group(P<0.05)and the glycogenolysis gene glycogen phosphorylase(pygl-1)mRNA increased in arsenite exposure group(P<0.05),presented a dose dependent manner.Compared with the control group,the expression of gluconeogenesis pathway gene phosphoenolpyruvate kinase(pck-1)mRNA decreased in arsenic exposure group(P<0.05)and fructose diphosphatase-1(fbp-1)mRNA increased only in 50μmol/L arsenic exposure group(P<0.05),and with the increase of sodium arsenite exposure,the level of pck-1 mRNA expression in nematodes showed a downward trend.Conclusion Chronic arsenic exposure may lead to glucose metabolism disorder in C.elegans by interfering with the expression of enzyme gene related to glucose metabolism.