Insulin Resistance Due to Unprocessed Insulin Proreceptor

未加工的胰岛素前受体导致胰岛素抵抗

• 批准号: 63480269
• 负责人: KOBAYASHI Masashi
• 金额: $ 4.42万
• 依托单位: Shiga University of Medical Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-63480269/
• 关键词: Insulin receptor disease; PCR; point mutation; Insulin receptor; Insulin resistance; インスリン抵抗性; 点突然変異; 糖尿病; インスリン誘導体; インスリン持抗性糖尿病; インスリンレセプター異常症; プロレセプター

We reported the siblings with severe insulin resistance because of unprocessed insulin proreceptors. To determine the structural abnormalities, we analyzed DNA sequence of the cleavage site by direct sequence method using PCR technique. The structural change of the cleavage site from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser due to G-T point mutation appeared to be the cause for failure to process the proreceptors. To study whether the mutation of insulin proreceptors at the cleavage site was responsible for unprocessed insulin proreceptors and to elucidate the structural and binding characteristics of the proreceptors, we performed transfection of cDNA with the mutation in COS 7 cells and examined the expressed insulin receptors. At 72 hours after transfection, insulin binding increased to the maximum in both cells transfected with normal cDNA or mutated cDNA, and they were 40 times and 14 times of control, non-transfected cells, respectively. Affinity cross-linking and surface labeling showed 135 kDa normal alpha subunit in cells transfected with normal cDNA and 210 kDa proreceptor in mutant cells. The proreceptors were cleaved by trypsin to alpha and beta subunit at the concentration of 0.025%. Auto-phosphorylation study showed decreased ^<32>P incorporation to proreceptors of cells transfected with mutated cDNA at both basal and insulin stimulated states without change in the sensitivity to insulin compared with cells transfected with normal cDNA. Competitive binding study with insulin, proinsulin and miniproinsulin showed that the proreceptors had lower relative affinity to proinsulin, but this characteristics disappeared by trypsin treatment. These results suggest that the mutation was the cause for unprocessed insulin proreceptors in the patients with insulin resistance, and that the expressed proreceptors of the COS 7 cells transfected with the mutated cDNA had similar binding specificity and sensitivity to trypsin.

我们报告了由于未加工的胰岛素前体而有严重胰岛素抵抗的兄弟姐妹。为了确定结构异常，我们采用PCR技术直接测序法分析了裂解位点的DNA序列。由于G-T点突变导致切割位点从Arg-Lys-Arg-Arg变为Arg-Lys-Arg-Ser，这可能是导致前体加工失败的原因。为了研究劈裂位点的胰岛素前受体突变是否导致了未加工的胰岛素前受体，并阐明了前受体的结构和结合特征，我们在COS 7细胞中转染了带有突变的cDNA，并检测了表达的胰岛素受体。转染后72小时，转染正常cDNA和突变cDNA的细胞中胰岛素结合量均达到最大值，分别是对照组和未转染细胞的40倍和14倍。亲和交联和表面标记显示，转染正常cDNA的细胞中正常α亚基为135 kDa，突变细胞中正常α亚基为210 kDa。在0.025%的浓度下，胰蛋白酶将前体切割成α和β亚基。自磷酸化研究表明，与转染正常cDNA的细胞相比，转染突变cDNA的细胞在基础状态和胰岛素刺激状态下，前受体中^<32>P的掺入减少，但对胰岛素的敏感性没有变化。与胰岛素、胰岛素原和迷你胰岛素原的竞争结合研究表明，前受体与胰岛素原的相对亲和力较低，但经胰蛋白酶处理后这种特性消失。这些结果表明，该突变是胰岛素抵抗患者胰岛素前受体未加工的原因，并且转染突变cDNA的COS 7细胞表达的胰岛素前受体对胰蛋白酶具有相似的结合特异性和敏感性。

项目成果

Kobayashi M, Sasaoka, T, Takata Y, et al.: "Unprocessed insulin proreceptors due to point mutation at the cleavage site." Diabetes Res Clin Prac. 7 :. 35-9, (1989)

Kobayashi M、Sasaoka、T、Takata Y 等人：“由于切割位点的点突变而导致未加工的胰岛素前受体。”

M.Kobayashi;T.Sasaoka;Y.Takata;et al.: Biochem Biophys Res Commun. 153. 657-663 (1988)

M.Kobayashi；T.Sasaoka；Y.Takata；等人：Biochem Biophys Res Commun。

M.Kobayashi他: "Unprocessed insulin proreceptors due to point mutation at the cleavage site" Diabetes Res.Clin.Practice. 7. S35-S39 (1989)

M.Kobayashi 等人：“由于切割位点的点突变而导致未加工的胰岛素前体”糖尿病研究临床实践 7. S35-S39 (1989)。

T.Sasaoka;M.Kobayashi;Y.Takata;et al.: Diabetes. 37. 1515-1523 (1988)

T.Sasaoka；M.Kobayashi；Y.Takata；等人：糖尿病。

Y.Takata;M.Kobayashi;et al.: "The International Symposium on Pathoqenesis ＆ Tretment of Type II Diabetes Mellitus" H.Abe;et al, 15-24 (1988)

Y.Takata；M.Kobayashi；等人：“II 型糖尿病发病机制和治疗国际研讨会” H.Abe；等人，15-24 (1988)