Generation of animal model of diabetes by disrupting the ADP-ribosyl cyclase (CD38) gene

通过破坏 ADP-核糖基环化酶 (CD38) 基因建立糖尿病动物模型

• 批准号: 08557009
• 负责人: KATO Ichiro
• 金额: $ 11.78万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08557009/
• 关键词: cyclic ADP-ribose; second messenger; knockout mouse; insulin; calcium ion; pancreatic beta cell; diabetes mellitus; transgenic mouse; グルコース; 糖負荷試験

1. We produced CD38 (+/-) mice by homologous recombination in ES cells and subsequent Cre-lox P recombination. By crosses between heterozygous mutants (+/-), homozygotes (-/-) were yielded in the F2 generation in a distribution following Mendelian rules ; hence CD38-/- mice seemed to survive fetal development normally.2.RT-PCR and Western blot analysis showed that there was no detectable CD38 mRNA and protein in pancreatic islets from CD38-/- mice, suggesting that the gene disruption resulted in a null mutation of CD38.3. As compared with CD38+/+ islet homogenates, the ADP-ribosyl cy clase, cADPR hydrolase and NAD+-glycohydrolase activities of CD38-/- islet homogenates were greatly reduced, indicating that CD38 is mainly responsible for the synthesis and hydrolysis of cADPR in pancreatic beta cells.4. By radioimmunoassay, the cADPR content in CD38+/+ islets was greatly increased by high glucose stimulation. Although some amounts of cADPR were detected in CD3 8-I- islets when incubated … More in low glucose, the cADPR content was not at all increased by high glucose stimulation.5. The glucose-stimulated [Ca2+]i rise in CD38-/- islets was much lower than that in CD38+/+ islets in the digital imaging of fura-2 fluorescence.6. Although there were no significant differences in insulin secretion between CD38+/+ and CD38-/- islets at 2.5 and 10 mM glucose, insulin secretion from CD38-/- islets at 20 and 30 mM glucose was more than 50% decreased compared with CD38+/+ islets.7. In glucose-tolerance test, At 30 and 60 min after glucose injection, CD38-/- mice had much higher glucose levels than CD38+/+ mice. In CD38-/- mice, serum insulin levels at 15 ruin after glucose injection were significantly lower than those of CD38+/+ mice.8. CD38-/- mice carrying the human CD38 transgene were generated. The human CD38 transgene ameliorated the glucose intolerance and the decreased insulin secretion.9. Overall results indicate that the CD38-/- mice are suitable animal model of noninsulin-dependent diabetes mellitus. In fact, we found CD38 missense mutation and autoantibodies against CD38 in noninsulin-dependent diabetic patients. Less

1.我们通过ES细胞中的同源重组和随后的Cre-lox P重组产生了CD 38（+/-）小鼠。通过杂合突变体（+/-）之间的杂交，在F2代中产生纯合子（-/-），其分布遵循孟德尔规则; RT-PCR和Western blot分析显示，CD 38-/-小鼠胰岛中未检测到CD 38 mRNA和蛋白，表明基因破坏导致CD 38.3的无效突变。与CD 38 +/+胰岛匀浆相比，CD 38-/-胰岛匀浆的ADP-核糖环化酶、cADPR水解酶和NAD+-糖水解酶活性均显著降低，表明CD 38主要负责胰腺β细胞中cADPR的合成和水解.放射免疫分析显示高糖刺激可显著增加CD 38 +/+胰岛cADPR含量。虽然在孵育的CD 3 8-I-胰岛中检测到一定量的cADPR， ...更多信息 在低糖条件下，高糖刺激对cADPR含量无明显影响.在fura-2荧光数字成像中，CD 38-/-胰岛中葡萄糖刺激的[Ca 2 +]i升高远低于CD 38 +/+胰岛.虽然在2.5和10 mM葡萄糖下CD 38 +/+和CD 38-/-胰岛之间的胰岛素分泌没有显著差异，但与CD 38 +/+胰岛相比，在20和30 mM葡萄糖下来自CD 38-/-胰岛的胰岛素分泌减少超过50%。在葡萄糖耐量试验中，注射葡萄糖后30和60 min，CD 38-/-小鼠的血糖水平明显高于CD 38 +/+小鼠。在CD 38-/-小鼠中，注射葡萄糖后15天的血清胰岛素水平显著低于CD 38 +/+小鼠.产生携带人⑶ 38转基因的⑶ 38-/-小鼠。人CD 38转基因改善了葡萄糖耐受不良和胰岛素分泌减少。总体结果表明，CD 38-/-小鼠是合适的非胰岛素依赖型糖尿病动物模型。事实上，我们在非胰岛素依赖型糖尿病患者中发现了CD 38错义突变和抗CD 38自身抗体。少

项目成果

Ichiro Kato: "CD38 disruption impairs glucose -induced increases in cyclic ADP-ribose, [Ca^<2->]i, and insulin secretion." Journal of Biological Chemistry. 274・4. 1869-1872 (1999)

Ichiro Kato：“CD38 破坏会损害葡萄糖诱导的循环 ADP-核糖、[Ca^<2->]i 和胰岛素分泌。”《生物化学杂志》274·4 (1999)。

阿部倫明: "新しいReg遺伝子(Reg IIIδ)の発見 : その構造決定とマウスRegファミリーの遺伝子地図の作製" 糖尿病. 42. in press (1999)

Michiaki Abe：“发现一个新的 Reg 基因（Reg IIIδ）：确定其结构并创建小鼠 Reg 家族的遗传图谱”糖尿病，出版 42。

Yagui, K.et al.: "A missense mutation in the CD38 gene, a novel factor for insulin secretion : association with Type II diabetes mellitus in Japanese subjects and evidence of abnormal function when expressed in vitro" Diabetologia. 41. 1024-1028 (1998)

Yagui, K.等人：“CD38 基因中的错义突变是胰岛素分泌的新因素：与日本受试者的 II 型糖尿病相关以及体外表达时功能异常的证据”Diabetologia。

阿部倫明: "新しいReg遺伝子(Reg IIIδ)の発見: その構造決定とマウスRegファミリーの遺伝子地図の作製" 糖尿病. 42(in press). (1999)

Michiaki Abe：“发现一个新的 Reg 基因（Reg IIIδ）：确定其结构并创建小鼠 Reg 家族的遗传图谱”糖尿病 42（出版中）。

高澤 伸: "糖尿病患者における抗REG I自己抗体の存在" 糖尿病. 42(in press). (1999)

Shin Takazawa：“糖尿病患者中存在抗 REG I 自身抗体”糖尿病 42（出版中）。