Studies on the beta cell differentiation signal in AR42J cells and in vivo effect on islet neogenesis in diabetic mice by betacellulin

我们试图阐明β细胞蛋白诱导AR42J细胞分化为胰岛素分泌细胞的机制，以及β细胞蛋白在体内对四氧嘧啶诱导的糖尿病小鼠胰岛新生和糖耐量的影响。在AR42J细胞中，重组人β细胞蛋白刺激诱导胰岛素免疫活性和胞浆中的β颗粒样分泌囊泡。erbB受体酪氨酸的磷酸化主要发生在不能直接与β细胞蛋白结合的erbB2中，提示在AR42J细胞向胰岛素产生的细胞内分化信号中，以异二聚体形式的erbB2的交叉激活是重要的。然而，我们在AR42J细胞表面没有检测到β细胞蛋白特异性受体或结合蛋白。β细胞蛋白主要在胎儿和成人胰腺中表达，我们发现该因子在胰岛的导管细胞和α细胞中产生。在四氧嘧啶诱导的糖尿病小鼠的胰腺中，胰岛新生主要发生在四氧嘧啶灌流的胰岛细胞缺失的部分。胰岛素转录因子IPF1/PDX-1在与新形成的胰岛样细胞团(ICCs)直接相关的导管细胞中检测到。在ICCs中，观察到对包括胰岛素在内的胰腺激素呈双阳性免疫反应的内分泌细胞，提示导管细胞在糖尿病小鼠胰岛新生中起重要作用。在此基础上，我们通过注射重组人β细胞蛋白对糖尿病小鼠体内的作用进行了研究。在用β细胞蛋白治疗的小鼠中，ICCs的数量增加，糖耐量减少。这些结果表明，正如在AR42J细胞中观察到的那样，β细胞蛋白可能是胰腺中的β细胞分化因子。

We tried to clarify the mechanism of differentiation of AR42J cells into insulin-secreting cells induced by betacellulin, and in vivo effects of betacellulin on the islet neogenesis and glucose intolerance in diabetic mice induced by selective perfusion of alloxan.In AR42J cells, stimulation with recombinant human betacellulin induced insulin immunoreactivlty and beta granule-like secretory vesicles in the cytoplasm.Phosphorylation of erbB receptor tyrosine was mainly occurred in erbB2 which could not bind directly to betacellulin, suggesting that crossactivation of erbB2 in the form of heterodimer is important for the intracellular differentiation signaling in AR42J cells into insulin-producing cells.However, it was difficult to clone the cells dominant negative for erbB2 by transfecting mutant form to confirm this result.We could not detect the betacellulin-specific receptor or binding protein on the cell surface of AR42J cells.Betacellulin was expressed predominantly in the fetal and adult pancreas, and we found this factor was produced in duct cells and alpha cells of islets.In the pancreas of diabetic mice induced by selective perfusion of alloxan, islet neogenesis was observed mainly in the alloxan-perfused beta cell-depleted segment.Expression of insulin transscription factor, IPF1/PDX-1 was detected in the duct cells directly associated with newly formed islet-like cell clusters (ICCs).In ICCs, endocrine cells with double positive immunoreactivities to pancreatic hormones including insulin were observed, suggesting duct cells are important for the islet neogenesis in diabetic pancreas.Based on these findings, we examined in vivo effect of betacellulin by injecting recombinant human betacellulin in these diabetic mice.In the mice treated with betacellulin, the number of ICCs was increased, and the glucose intolerance was ameliorated.These results suggested betacellulin, as observed in AR42J cells, may act as beta cell differentiation factor in the pancreas.