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

本研究旨在阐明β细胞素诱导AR 42 J细胞分化为胰岛素分泌细胞的机制，以及β细胞素对四氧嘧啶诱导的糖尿病小鼠胰岛新生和糖耐量的影响。用重组人β细胞素刺激诱导胰岛素免疫反应性和β颗粒，erbB受体酪氨酸的磷酸化主要发生在erbB 2上，erbB 2不能直接与β细胞素结合，这表明异二聚体形式的erbB 2的交叉激活对于AR 42 J细胞向胰岛素产生细胞的细胞内分化信号传导是重要的。然而，我们在AR 42 J细胞表面未检测到β细胞素特异性受体或结合蛋白。β细胞素主要在胎儿和成人胰腺中表达，我们发现这种因子在导管细胞和胰岛α细胞中产生。在选择性灌注四氧嘧啶诱导的糖尿病小鼠胰腺中，胰岛新生主要发生在四氧嘧啶灌注的β细胞缺失段。在与新形成的胰岛样细胞簇（ICC）直接相关的导管细胞中检测到胰岛素转录因子IPF 1/PDX-1的表达。在ICC中，结果表明，糖尿病小鼠胰岛内分泌细胞对胰岛素等胰腺激素呈双阳性反应，提示导管细胞在胰岛新生中起重要作用，在此基础上，我们观察了β细胞素对糖尿病小鼠胰岛内分泌细胞的影响，结果表明，β细胞素可使糖尿病小鼠胰岛内分泌细胞数量增加，这些结果表明，在AR 42 J细胞中观察到的β细胞素可能在胰腺中作为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.