Molecular mechanisms of insulin signal transduction and diabetes mellitus

胰岛素信号转导与糖尿病的分子机制

• 批准号: 14370045
• 负责人: EBINA Yousuke
• 金额: $ 8.9万
• 依托单位: The University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370045/
• 关键词: Insulin signalling; APS; KO mouse; diabetes; Serum insulin receptor; 糖尿病; 血中マーカー; インスリン作用; GLUT4トランスロケーション; グルコース取り込み; Aktキナーゼ

APS-deficient (APS(-/-)) mice were used to investigate its function in vivo. The blood glucose-lowering effect of insulin, as assessed by the intraperitoneal insulin tolerance test, was increased in APS(-/-) mice. APS(-/-) mice showed an increase in the whole-body glucose infusion rate as assessed by the hyperinsulinemic-euglycemic clamp test. These findings indicated that APS(-/-) mice exhibited increased sensitivity to insulinUsing the expression vector of the truncated human insulin receptor (hIR), we have constructed a stable Chinese hamster ovary (CHO) cell line which secretes the His-tagged α subunit (insulin-binding domain) of hIR into medium. To examine characteristics of the His-tagged hIRα, we purified the protein secreted from the CHO cells. The His-tagged hIRα was glycosylated and processed a dimer. The molecule bound insulin with an affinity similar to that of the intact hIR. The His-tagged full length of hIR was autophosphorylated by insulin stimulation in CHO cells. Injection of the purified His-tagged hIRα into veins of mice increased in the concentration of blood glucose within 30 min. The intraperitoneal glucose tolerance test (ipGTT) done after injection of the purified His-tagged hIRα showed evidence of a marked hyperglycemia. These findings provide direct evidence that the presence of hlRα in the blood stream inhibits insulin actions by binding with plasma insulin

用APS基因缺陷(APS(-/-))小鼠研究其体内功能。在APS(-/-)小鼠中，通过腹膜胰岛素耐量试验评估，胰岛素的降糖作用增强。通过高胰岛素-正常血糖钳夹试验评估，APS(-/-)小鼠的全身葡萄糖输注率增加。利用截短人胰岛素受体(HIR)的表达载体，我们构建了一个稳定的中国仓鼠卵巢(CHO)细胞系，该细胞系能分泌His标记的His标记的α亚单位(胰岛素结合域)到培养液中。为了检测His标记的HIRα的特性，我们纯化了CHO细胞分泌的蛋白质。His标记的hirα被糖基化并加工成二聚体。该分子与胰岛素结合的亲和力与完整的HIR相似。His标记的全长HIR在CHO细胞中被胰岛素刺激而自动磷酸化。静脉注射纯化的His标记的HIRα可在30min内升高小鼠的血糖浓度。注射纯化的His标记的hIRα后进行的腹膜糖耐量试验(IpGTT)显示有明显的高血糖。这些发现提供了直接的证据，证明血液中hlrα的存在通过与血浆胰岛素结合来抑制胰岛素的作用。

项目成果

Toshiyuki Obata et al.: "Use of RNA-interference-mediated gene silencing and adenoviral overexpression to Elucidate the roles of AKT/PKB-isoforms in insulin actions"J.Biol.Chem.. 278. 28312-28323 (2003)

Toshiyuki Obata 等人：“使用 RNA 干扰介导的基因沉默和腺病毒过表达来阐明 AKT/PKB 亚型在胰岛素作用中的作用”J.Biol.Chem.. 278. 28312-28323 (2003)

Yoshiko Kanezaki et al.: "Injection of the insulin receptor alpfa subunit increases blood glucose levels in mice"Biochem.Biophys.Res.Commun.. 309, 30. 572-577 (2003)

Yoshiko Kanezaki 等人：“注射胰岛素受体 apfa 亚基会增加小鼠的血糖水平”Biochem.Biophys.Res.Commun.. 309, 30. 572-577 (2003)

Asako Minami et al.: "Increased insulin sensitivity and hypoinsulinemia in APS knockout mice"Diabetes. 52. 2657-2665 (2003)

Asako Minami 等人：“APS 基因敲除小鼠的胰岛素敏感性增加和低胰岛素血症”糖尿病。

Asako Minami: "Increased insulin sensitivity and hypoinsulinemia in APS knockout mice"Diabetes. 52. 2657-2665 (2003)

Asako Minami：“APS 基因敲除小鼠的胰岛素敏感性增加和低胰岛素血症”糖尿病。

Yoshiko Kanezaki et al.: "KATP Channel Knockout Mice with transgenic Mice Expressing a Dominant-Negative Form of Human Insulin receptor have Glucose Intolerance but not Diabetes"Endocrine J.. (in press).

Yoshiko Kanezaki 等人：“KATP 通道敲除小鼠与表达人胰岛素受体显性阴性形式的转基因小鼠有葡萄糖不耐受，但没有糖尿病”Endocrine J..（出版中）。