摘要目的 探讨血管紧张素Ⅱ对小鼠胰岛β细胞内胰岛素信号通路的影响及可能机制.方法 小鼠胰岛素分泌细胞株NIT-1以低糖培养基和无血清培养液培养后分为6组:血管紧张素Ⅱ组(A组)、胰岛素组(B组)、血管紧张素Ⅱ+胰岛素组(C组)、血管紧张素Ⅱ+胰岛素+沙拉新组(D组)、血管紧张素Ⅱ+胰岛素+二联苯碘组(E组)、对照组(F组).应用Western blot检测酪氨酸磷酸化胰岛素受体β亚单位、酪氨酸磷酸化胰岛素受体底物-1、丝氨酸磷酸化胰岛素受体底物-1及P47phox蛋白表达水平,采用流式细胞仪检测细胞内H2O2水平,选用逆转录-聚合酶链反应检测胰岛素mRNA水平.组间比较采用方差分析,两两比较采用LSD检验.结果 A组丝氨酸磷酸化胰岛素受体底物-1较F组显著升高(分别为1.18±0.10和0.59±0.11,LSD检验,P＜0.01).与B组比较,C组酪氨酸磷酸化胰岛素受体β亚单位(分别为1.22±0.26和1.95±0.19)、酪氨酸磷酸化胰岛素受体底物-1(分别为0.74±0.18和1.25±0.23)明显降低(均P＜0.01),丝氨酸磷酸化胰岛素受体底物-1显著增加(分别为1.11±0.17和0.62±0.10,P＜0.01).D组、E组酪氨酸磷酸化胰岛素受体β亚单位、酪氨酸磷酸化胰岛素受体底物-1较C组升高,丝氨酸磷酸化胰岛素受体底物-1 下降.A组、C组细胞内H2O2显著高于F组(分别为44.2±2.2、41.0±5.0和28.6±1.7,均P＜0.01),D组、E组低于C组(分别为30.2±1.7、30.8±2.1和41.0±5.0,均P＜0.01).A组、C组P47 phox水平明显高于F组(分别为1.62±0.17、1.59±0.19和0.89±0.12,均P＜0.01),D组、E组P47 phox水平显著低于C组(分别为1.09±0.16、30.8±2.1和1.59±0.19,均P＜0.01).就胰岛素mRNA表达水平而言,B组显著高于F组(分别为0.90±0.15和0.60±0.19,P＜0.01),C组低于B组(分别为0.62±0.19和0.90±0.15,P＜0.01),D组、E组明显高于C组(分别为0.80±0.17、0.82±0.19和0.62±0.19,均P＜0.05).结论 血管紧张素Ⅱ可增加小鼠NIT-1细胞株丝氨酸磷酸化胰岛素受体底物-1水平,降低胰岛素刺激的酪氨酸磷酸化胰岛素受体β亚单位、酪氨酸磷酸化胰岛素受体底物-1水平,此效应通过活化烟酰胺腺嘌呤二核苷酸磷酸酶导致氧化应激增加而介导.

abstractsObjective To evaluate the effects of angiotensin Ⅱ on insulin signaling pathway in insulin-secreting NIT-1 cells in mice. Methods NIT-1 eells were cultured in the modified DMED,and then in the serum-free DMEM before assigned to the following 6 groups: angiotensin Ⅱ group ( group A ),insulin group ( group B) ,angiotensin Ⅱ + insulin group ( group C ) ,angioteusin Ⅱ +insulin + saralasin group (group D ),angiotensin Ⅱ + insulin + Diphenyleneiodonium group (group E ) and control group (group F). Expression of insulin receptor tyrosine phosphorylation (IR-β-Tyr) ,tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1-Tyr),serine phosphorylation of insulin receptor substrate-1 (IRS-1-Ser),and P47 phox were evaluated by Western blot Level of H2O2 was detected by flow cytometry.Expression of insulin mRNA was evaluated by reverse transcript-polymerase chain reaction. One-way analysis of variance (ANOVA) and LSD test were used for data analysis. Results Expression of IRS-1-Ser was higher in the group A than group F. The levels of IR-β-Tyr (1.22 ±0.26 vs 1.95 ±0. 19,LSD test,P＜0. 01 ) and IRS-1-Tyr (0. 74 ±0. 18 vs 1.25 ±0. 23,P ＜0. 01 ) in group C were lower than those in group B,whereas the level of IRS-Ser was higher ( 1. 11 ± 0. 17 vs 0. 62 ± 0. 10,P ＜ 0. 01 ). The expressions of IR-β-Tyr and IRS-1-Tyr in groups D and E were significantly increased when compared with group C,and the level of IRS-1-Ser changed adversely. The levels of H2O2 and P47 phox in groups A and C were significantly increased when compared with group F,and those in groups D and E were lower in comparison with group C. The expression of insulin mRNA was significantly increased in groups D and E than group C ( group D vs group C: 0. 80 ± 0. 17 vs 0. 62 ± 0. 19,P ＜ 0. 05; group Evs group C: 0. 82 ± 0. 19 vs 0. 62 ±0. 19,P ＜ 0. 01 ). Conclusions Angiotensin Ⅱ could increase the expression of IRS-1-Ser and inhibit insulin-stimulated IR-β-Tyr and IRS-1-Tyr in NIT-1 cells. Oxidative stress induced by nicotinamide adenine dinucleotide phosphate oxidase may play an important role in the effect of angiotensin Ⅱ in NIT-1 cells.