Glucose regulated production of human insulin in rat hepatocytes
Glucose regulated production of human insulin in rat hepatocytes
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DOI：
10.1038/sj.gt.3301076
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发表时间：
2000-02-01
期刊：
影响因子：
5.1
通讯作者：
Phillips, LS
中科院分区：
文献类型：
--
作者：
Thulé, PM;Liu, J;Phillips, LS
胰岛素基因治疗需要胰岛素分泌与代谢需求相结合。为此，我们开发了一种胰岛素转基因，其转录受葡萄糖刺激并受胰岛素抑制。通过将大鼠 L-丙酮酸激酶 (L-PK) 基因的葡萄糖响应元件 (GIRE) 插入胰岛素敏感、肝脏特异性、大鼠胰岛素样生长因子结合蛋白 1 (IGFBP-1) 启动子中，构建了葡萄糖和胰岛素敏感启动子。在原代培养的大鼠肝细胞中，葡萄糖（5至25 mM）刺激和胰岛素（10（-10）至10（-7）M）抑制由这些启动子驱动的荧光素酶表达。使用人胰岛素原 cDNA (2xfur) 证明了转染的肝细胞分泌成熟、具有生物活性的胰岛素的能力，该 cDNA 经修饰以允许通过内源内肽酶活性进行蛋白质加工。由产生胰岛素的肝细胞调节的培养基含有大于300 mu U/ml的免疫反应性胰岛素，而抗胰岛素免疫沉淀物的变性SDS-PAGE显示具有胰岛素A和B链迁移率的条带。转染试验证明了肝细胞产生的胰岛素的生物活性，其中由产生胰岛素的肝细胞调节的培养基发挥了与10(-7) M胰岛素相似的效果。然后，我们将葡萄糖和胰岛素敏感的启动子与修饰的人胰岛素原 cDNA 结合起来，创建代谢敏感的胰岛素转基因 ((GIRE)(3)BP-1 2xfur)。在用该构建体稳定转染的 H4IIE 肝癌细胞和正常 mt 肝细胞 (GIRE)(3)BP-1 2xfur 介导的胰岛素分泌中，响应葡萄糖的刺激而增加。此外，还证明了响应葡萄糖暴露减少而减少胰岛素产生的能力。我们得出的结论是，使用这些葡萄糖和胰岛素敏感构建体对胰岛素产生的转录调节满足在胰岛素基因治疗的啮齿动物模型中应用的要求。
Insulin gene therapy requires that insulin secretion be coupled to metabolic requirements. To this end, we have developed an insulin transgene whose transcription is stimulated by glucose and inhibited by insulin. Glucose- and insulin-sensitive promoters were constructed by inserting glucose-responsive elements (GIREs) from the rat L-pyruvate kinase (L-PK) gene into the insulin-sensitive, liver-specific, rat insulin-like growth factor binding protein-1 (IGFBP-1) promoter. Glucose (5 to 25 mM) stimulated and insulin (10(-10) to 10(-7) M) inhibited luciferase expression driven by these promoters in primary cultured rat hepatocytes. The capacity of transfected hepatocytes to secrete mature, biologically active insulin was demonstrated using a human proinsulin cDNA (2xfur), modified to allow protein processing by endogenous endopeptidase activity. Medium conditioned by insulin-producing hepatocytes contained greater than 300 mu U/ml immunoreactive insulin, while denaturing SDS-PAGE of an anti-insulin immunoprecipitate revealed bands with the mobilities of insulin A, and B chains. Biological activity of hepatocyte-produced insulin was demonstrated in a transfection assay, in which medium conditioned by insulin-producing hepatocytes exerted an effect similar to 10(-7) M insulin. We then combined the glucose- and insulin-sensitive promoter with the modified human proinsulin cDNA to create a metabolically sensitive insulin transgene ((GIRE)(3)BP-1 2xfur). In both H4IIE hepatoma cells stably transfected with this construct, and normal mt hepatocytes (GIRE)(3)BP-1 2xfur-mediated insulin secretion increased in response to stimulation by glucose. Moreover a capacity to decrease insulin production in response to diminishing glucose exposure was also demonstrated We conclude that the transcriptional regulation of insulin production using these glucose- and insulin-sensitive constructs meets the requirements for application in a rodent model of insulin gene therapy.