Yang Shumin, Fransson Ulrika, Fagerhus Lillian, Holst Lena Stenson, Hohmeier Hans E, Renström Erik, Mulder Hindrik
Department of Cell and Molecular Biology, Lund University, SE-221 84 Lund, Sweden.
Mol Endocrinol. 2004 Sep;18(9):2312-20. doi: 10.1210/me.2004-0148. Epub 2004 May 27.
In type 2 diabetes, beta-cells become glucose unresponsive, contributing to hyperglycemia. To address this problem, we recently created clonal insulin-producing cell lines from the INS-1 insulinoma line, which exhibit glucose responsiveness ranging from poor to robust. Here, mechanisms that determine secretory performance were identified by functionally comparing glucose-responsive 832/13 beta-cells with glucose-unresponsive 832/2 beta-cells. Thus, insulin secretion from 832/13 cells maximally rose 8-fold in response to glucose, whereas 832/2 cells responded only 1.5-fold. Insulin content in both lines was similar, indicating that differences in stimulus-secretion coupling account for the differential secretory performance. Forskolin or isobutylmethylxanthine markedly enhanced insulin secretion from 832/13 but not from 832/2 cells, suggesting that cAMP is essential for the enhanced secretory performance of 832/13 cells. Indeed, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, rp-isomer (Rp-8-Br-cAMPS) an inhibitor of protein kinase A (PKA), inhibited insulin secretion in response to glucose with or without forskolin. Interestingly, whereas forskolin markedly increased cAMP in 832/2 cells, 832/13 cells exhibited only a marginal rise in cAMP. This suggests that 832/13 cells are more sensitive to cAMP. Indeed, the cAMP-induced exocytotic response in patch-clamped 832/13 cells was 2-fold greater than in 832/2 cells. Furthermore, immunoblotting revealed that expression of the catalytic subunit of PKA was 2-fold higher in 832/13 cells. Moreover, when the regulatory subunit of PKA was overexpressed in 832/13 cells, to reduce the level of unbound and catalytically active kinase, insulin secretion and PKA activity were blunted. Our findings show that cAMP-PKA signaling correlates with secretory performance in beta-cells.
在2型糖尿病中，β细胞对葡萄糖不再产生反应，从而导致高血糖症。为了解决这个问题，我们最近从INS-1胰岛素瘤细胞系中创建了克隆性胰岛素分泌细胞系，这些细胞系对葡萄糖的反应能力从较弱到较强不等。在这里，通过对葡萄糖反应性的832/13β细胞和葡萄糖无反应性的832/2β细胞进行功能比较，确定了决定分泌性能的机制。因此，832/13细胞的胰岛素分泌在葡萄糖刺激下最大可增加8倍，而832/2细胞仅增加1.5倍。两株细胞中的胰岛素含量相似，这表明刺激-分泌偶联的差异是分泌性能不同的原因。福斯可林或异丁基甲基黄嘌呤可显著增强832/13细胞的胰岛素分泌，但对832/2细胞无此作用，这表明cAMP对832/13细胞增强的分泌性能至关重要。事实上，蛋白激酶A（PKA）的抑制剂8-溴腺苷-3',5'-环磷酸硫酯，rp-异构体（Rp-8-Br-cAMPS）可抑制有无福斯可林时葡萄糖刺激引起的胰岛素分泌。有趣的是，虽然福斯可林可显著增加832/2细胞中的cAMP，但832/13细胞中的cAMP仅略有升高。这表明832/13细胞对cAMP更敏感。事实上，膜片钳记录的832/13细胞中cAMP诱导的胞吐反应比832/2细胞大2倍。此外，免疫印迹显示832/13细胞中PKA催化亚基的表达高2倍。此外，当PKA调节亚基在832/13细胞中过表达以降低未结合和具有催化活性的激酶水平时，胰岛素分泌和PKA活性均减弱。我们的研究结果表明，cAMP-PKA信号传导与β细胞的分泌性能相关。
