Regeneration of pancreatic beta-cells that secret insulin in response to glucose stimulation

催乳素调节元件结合蛋白（PREB）已被确定为调节大鼠垂体前叶催乳素启动子活性的因子。PREB不仅位于垂体前叶，也位于胰腺，但其在胰腺中的作用尚不清楚。因此，我们研究了PREB在胰岛素基因表达中的作用。采用报告基因分析和EMSA技术分析PREB对胰岛素基因转录的影响。在表达或敲低PREB的细胞中，测定胰岛素表达和分泌。PREB主要定位于大鼠胰岛β细胞及其细胞系INS-1的细胞核内。INS-1细胞的核提取物含有被PREB抗血清识别的物质。此外，该核提取物含有胰岛素启动子结合活性，其在EMSA研究中被PREB抗血清超移位。在INS-1细胞中，PREB和胰岛素启动子的共表达诱导了后者的活性。向细胞中添加葡萄糖增加PREB表达。胰岛素启动子的缺失分析表明，胰岛素启动子中的葡萄糖响应顺式元件A3介导PREB的转录作用。此外，合成的PREB通过EMSA结合A3元件，而胰岛素启动子中该基序的突变体消除了PREB的作用。此外，表达或敲低PREB的细胞分别表现出增加或减少的胰岛素表达。这些结果表明，PREB可能有助于调节胰岛素基因转录和胰岛素分泌响应葡萄糖刺激。

PREB (prolactin regulatory element binding) protein has been identified as a factor that regulates prolactin promoter activity in rat anterior pituitary. PREB is located not only in the anterior pituitary but also in pancreas, however its role in pancreas is not known. Therefore, we examined the role of PREB in insulin gene expression. To analyze the effects of PREB on insulin gene transcription, we employed the reporter gene assay and EMSA. In the cells expressing or knocked down the PREB, insulin expression and secretion were determined. PREB was located mainly in nuclei of rat pancreatic β-cells and its cell line, INS-1. A nuclear extract of INS-1 cells contained material that was recognized by PREB antiserum. Furthermore, this nuclear extract contained insulin promoter binding activity that was super-shifted by PREB antiserum in EMSA studies. In the INS-1 cells, the co-expression of PREB and the insulin promoter induced activity of the latter. The addition of glucose to the cells increased PREB expression. Deletional analysis of the insulin promoter showed that A3, a glucose-responsive cis-element in the insulin promoter mediated the transcriptional effect of PREB. Additionally, synthesized PREB bound the A3 element by EMSA, while a mutant of this motif in the insulin promoter abrogated the effect of PREB. Furthermore, cells expressing or knock-down PREB exhibited increased or decreased insulin expression, respectively. These results demonstrate that PREB may contribute to the regulation of insulin gene transcription and insulin secretion in response to glucose stimulation.