Biomedical Research Service, John D. Dingell VA Medical Center, Detroit, MI, USA.
Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI, USA.
Cell Physiol Biochem. 2020 Dec 12;54(6):1218-1230. doi: 10.33594/000000310.
BACKGROUND/AIMS: Despite the published evidence implicating phosphoinositide 3-kinase (PI3-kinase) in the regulation of islet function, limited information is available on the putative contributory roles of its downstream signaling steps, including the phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-Rex1) signaling pathway in the islet β-cell. Therefore, we investigated potential roles for P-Rex1 in glucose-stimulated Rac1 activation and insulin secretion in insulin-secreting (INS-1 832/13) β-cells.
Glucose-stimulated Insulin secretion (GSIS) was quantified by ELISA. Expression of endogenous P-Rex1 and RhoG was suppressed by siRNA transfection using the DharmaFect1 reagent. Total membrane and cytosolic fractions were isolated using the Mem-PER Plus Membrane Extraction Kit. The degree of activation of Rac1 was determined by the pull-down assay.
P-Rex1 is expressed in INS-1 832/13 cells, normal rat islets and human islets. siRNA-mediated knockdown of P-Rex1 attenuated glucose-induced Rac1 activation, membrane association and insulin secretion. RhoG, which has been implicated in PI3-kinase-mediated Rac1 activation in other cell types, appears not to contribute to GSIS since the siRNA-mediated knockdown of RhoG failed to exert significant effects on GSIS. LY294002, a known inhibitor of PI3-kinase, potentiated GSIS without affecting glucose-induced Rac1 activation.
Based on these findings, we conclude that P-Rex1 plays a novel regulatory role in glucose-induced Rac1 activation and insulin secretion.
背景/目的：尽管已有文献表明磷酸肌醇 3-激酶（PI3-kinase）参与了胰岛功能的调节，但关于其下游信号通路（包括磷酸肌醇-3,4,5-三磷酸依赖性 Rac 交换因子 1（P-Rex1）信号通路）在胰岛 β 细胞中的潜在作用的信息有限。因此，我们研究了 P-Rex1 在葡萄糖刺激的 Rac1 激活和胰岛素分泌中的潜在作用在胰岛素分泌细胞（INS-1 832/13）β 细胞中。
通过 ELISA 定量测定葡萄糖刺激的胰岛素分泌（GSIS）。使用 DharmaFect1 试剂转染 siRNA 抑制内源性 P-Rex1 和 RhoG 的表达。使用 Mem-PER Plus 膜提取试剂盒分离总膜和胞质部分。通过下拉测定法测定 Rac1 的激活程度。
P-Rex1 在 INS-1 832/13 细胞、正常大鼠胰岛和人胰岛中表达。siRNA 介导的 P-Rex1 敲低减弱了葡萄糖诱导的 Rac1 激活、膜结合和胰岛素分泌。RhoG 已被证明在其他细胞类型中参与 PI3-kinase 介导的 Rac1 激活，但似乎不会对 GSIS 产生重大影响，因为 RhoG 的 siRNA 介导的敲低对 GSIS 没有显著影响。LY294002 是一种已知的 PI3-kinase 抑制剂，增强了 GSIS，而不影响葡萄糖诱导的 Rac1 激活。
基于这些发现，我们得出结论，P-Rex1 在葡萄糖诱导的 Rac1 激活和胰岛素分泌中发挥新的调节作用。
