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. 2021 Apr 14;55(2):180-192. doi: 10.33594/000000354.
BACKGROUND/AIMS: Published evidence suggests regulatory roles for small G proteins (Cdc42 and Rac1) in glucose-stimulated insulin secretion (GSIS) from pancreatic beta-cells. More recent evidence suggests novel roles for these G proteins, specifically Rac1, in the induction of metabolic dysfunction of the islet beta-cell under the duress of a variety of stress conditions. However, potential upstream regulators of sustained activation of Rac1 have not been identified in the beta-cell. Recent studies in other cell types have identified RhoG, a small G protein, as an upstream regulator of Rac1 under specific experimental conditions. Herein, we examined putative roles for RhoG in islet beta-cell dysregulation induced by glucotoxic conditions.
Expression of RhoG or GDIγ was suppressed by siRNA transfection using the DharmaFect1 reagent. Subcellular fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagent kit. The degree of activation of Rac1 was assessed using a pull-down assay kit. Extent of cell death was quantified using a Cell Death Detection ELISA kit.
RhoG is expressed in human islets, rat islets, and clonal INS-1 832/13 cells. siRNA-RhoG markedly attenuated sustained activation of Rac1 and caspase-3 in INS-1 832/13 cells exposed to hyperglycemic conditions (20 mM; 24 hours). In a manner akin to Rac1, which has been shown to translocate to the nuclear fraction to induce beta-cell dysfunction under metabolic stress, a significant increase in the association of RhoG with the nuclear fraction was observed in beta-cells under the duress of metabolic stress. Interestingly, GDIγ, a known regulator of RhoG, remained associated with non-nuclear fraction under conditions RhoG and Rac1 translocated to the membrane. Lastly, siRNA-RhoG modestly attenuated pancreatic beta-cell demise induced by high glucose exposure conditions, but such an effect was not statistically significant.
Based on these data we conclude that RhoG-Rac1 signaling module plays critical regulatory roles in promoting mitochondrial dysfunction (caspase-3 activation) of the islet beta cell under metabolic stress.
背景/目的：已有研究表明，小分子 G 蛋白（Cdc42 和 Rac1）在胰腺β细胞的葡萄糖刺激胰岛素分泌（GSIS）中发挥调节作用。最近的研究表明，在各种应激条件下，这些 G 蛋白（特别是 Rac1）在胰岛β细胞代谢功能障碍的诱导中发挥了新的作用。然而，在β细胞中，尚未发现 Rac1 持续激活的潜在上游调节剂。最近在其他细胞类型中的研究表明，在特定实验条件下，小分子 G 蛋白 RhoG 是 Rac1 的上游调节剂。在此，我们研究了 RhoG 在葡萄糖毒性诱导的胰岛β细胞失调中的潜在作用。
使用 DharmaFect1 试剂通过 siRNA 转染抑制 RhoG 或 GDIγ 的表达。使用 NE-PER 核和细胞质提取试剂试剂盒分离亚细胞级分。使用下拉测定试剂盒评估 Rac1 的激活程度。使用细胞死亡检测 ELISA 试剂盒定量细胞死亡程度。
RhoG 在人胰岛、大鼠胰岛和克隆 INS-1 832/13 细胞中表达。siRNA-RhoG 显著减弱了高糖条件（20 mM；24 小时）下 INS-1 832/13 细胞中 Rac1 的持续激活和半胱天冬酶-3的激活。与在代谢应激下已被证明易位到核部分以诱导β细胞功能障碍的 Rac1 类似，在代谢应激下，β细胞中 RhoG 与核部分的结合显著增加。有趣的是，在 RhoG 和 Rac1 易位到膜的情况下，GDIγ，一种已知的 RhoG 调节剂，仍与非核部分结合。最后，siRNA-RhoG 适度减弱了高糖暴露条件下胰岛β细胞的死亡，但这种作用没有统计学意义。
基于这些数据，我们得出结论，RhoG-Rac1 信号模块在代谢应激下促进胰岛β细胞的线粒体功能障碍（半胱天冬酶-3 激活）中发挥关键调节作用。
