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目的:探讨中心粒周蛋白(pericentrin,PCNT)对胰岛素双相分泌的调节作用及其机制。方法:构建在小鼠胰岛β细胞中PCNT表达减少的转基因鼠(△PCNTβ小鼠),检测△PCNTβ小鼠与正常对照小鼠在给予糖耐量试验后第一时相和第二时相血糖和胰岛素分泌情况。检测两组小鼠胰岛内PCNT、胰岛素、纤维肌动蛋白(F-actin)变化情况,以及相关蛋白表达情况。结果:Western blot和RT-PCR显示△PCNTβ小鼠胰尾组织PCNT表达较对照组明显减低,免疫荧光提示△PCNTβ小鼠胰岛内PCNT、胰岛素表达较对照组明显减低。行腹腔注射葡萄糖耐量试验(Intraperitoneal glucose tolerance tests,IPGTT)△PCNTβ小鼠第一时相血糖曲线下面积(Quantification of area under the curve,AUC)显著高于对照组,第二时相血糖AUC两组小鼠间无统计学差异。△PCNTβ小鼠空腹胰岛素水平与对照组比较明显升高,葡萄糖刺激胰岛素分泌(Glucose stimulated insulin secretion,GSIS)后15min胰岛素增加值显著低于对照组,30 min和120 min时胰岛素水平与对照组无显著差异。Western blot显示△PCNTβ小鼠与对照组比较F-actin表达明显减低,ERK、p-ERK表达明显升高。RT-PCR测定△PCNTβ小鼠与对照组比较ETV4表达显著升高。免疫荧光提示△PCNTβ小鼠胰岛内F-actin和突触融合蛋白4(Syntaxin4,Syn-4)表达较对照组明显减低。结论:抑制小鼠胰岛β细胞内PCNT表达后,其通过抑制F-actin和Syn-4表达影响胰岛素分泌,导致空腹时胰岛素过度分泌和第一时相胰岛素分泌受损。
Objective: To investigate the regulatory effect of pericentrin (PCNT) on biphasic insulin secretion and its mechanism. Methods: The transgenic mice (△ PCNTβ mice) with reduced PCNT expression in islet β cells of mice were constructed. The △ PCNTβ mice and normal control mice were given glucose tolerance test after the first phase and second phase glucose Insulin secretion. The changes of PCNT, insulin and F-actin in the islets of the two groups of mice were detected, and the expression of related proteins were detected. Results: Western blot and RT-PCR showed that the expression of PCNT in pancreatic tails of △ PCNTβ mice was significantly lower than that in control group. Immunofluorescence indicated that the expression of PCNT and insulin in △ PCNTβ mice was significantly lower than that in control group. The Quantification of area under the curve (AUC) of △ PCNTβ in intraperitoneal glucose tolerance tests (IPGTT) group was significantly higher than that in control group, and the second phase of blood glucose AUC There was no significant difference between mice. The fasting insulin level in △ PCNTβ mice was significantly higher than that in the control group. The insulin added value at 15 min after glucose-stimulated insulin secretion (GSIS) was significantly lower than that in the control group. Insulin levels at 30 min and 120 min were significantly lower than those in the control group Significant differences. Western blot showed that the expression of F-actin was significantly decreased and the expression of ERK and p-ERK was significantly increased in △ PCNTβ mice compared with the control group. The expression of ETV4 in △ PCNTβ mice was significantly higher than that in the control group by RT-PCR. Immunofluorescence indicated that the expression of F-actin and Syn-4 in the islet of △ PCNTβ mice was significantly lower than that of the control group. CONCLUSION: Inhibition of PCNT expression in pancreatic β-cells inhibits insulin secretion by inhibiting the expression of F-actin and Syn-4, resulting in impaired fasting insulin and impaired first phase insulin secretion.