Dept. of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State Univ., Detroit, MI 48201, USA.
Am J Physiol Regul Integr Comp Physiol. 2011 Mar;300(3):R756-62. doi: 10.1152/ajpregu.00786.2010. Epub 2011 Jan 12.
Recent evidence suggests that an acute increase in the generation of phagocyte-like NADPH-oxidase (Nox)-mediated reactive oxygen species (ROS) may be necessary for glucose-stimulated insulin secretion. Using rat islets and INS 832/13 cells, we tested the hypothesis that activation of specific G proteins is necessary for nutrient-mediated intracellular generation of ROS. Stimulation of β-cells with glucose or a mixture of mitochondrial fuels (mono-methylsuccinate plus α-ketoisocaproic acid) markedly elevated intracellular accumulation of ROS, which was attenuated by selective inhibitors of Nox (e.g., apocynin or diphenyleneiodonium chloride) or short interfering RNA-mediated knockdown of p47(phox), one of the subunits of Nox. Selective inhibitors of protein prenylation (FTI-277 or GGTI-2147) markedly inhibited nutrient-induced ROS generation, suggesting that activation of one (or more) prenylated small G proteins and/or γ-subunits of trimeric G proteins is involved in this signaling axis. Depletion of endogenous GTP levels with mycophenolic acid significantly reduced glucose-induced activation of Rac1 and ROS generation in these cells. Other immunosuppressants, like cyclosporine A or rapamycin, which do not deplete endogenous GTP levels, failed to affect glucose-induced ROS generation, suggesting that endogenous GTP is necessary for glucose-induced Nox activation and ROS generation. Treatment of INS 832/13 cells or rat islets with pertussis toxin (Ptx), which ADP ribosylates and inhibits inhibitory class of trimeric G proteins (i.e., G(i) or G(o)), significantly attenuated glucose-induced ROS generation in these cells, implicating activation of a Ptx-sensitive G protein in these signaling cascade. Together, our findings suggest a prenylated Ptx-sensitive signaling step couples Rac1 activation in the signaling steps necessary for glucose-mediated generation of ROS in the pancreatic β-cells.
最近的证据表明，吞噬细胞样 NADPH 氧化酶（Nox）介导的活性氧（ROS）的急性增加可能是葡萄糖刺激胰岛素分泌所必需的。使用大鼠胰岛和 INS 832/13 细胞，我们检验了这样一个假设，即激活特定的 G 蛋白对于营养介导的细胞内 ROS 生成是必要的。用葡萄糖或线粒体燃料混合物（单甲基琥珀酸加 α-酮异己酸）刺激β细胞，明显增加了细胞内 ROS 的积累，这一作用被 Nox 的选择性抑制剂（如 apocynin 或二苯并碘化物）或 p47(phox)（Nox 的一个亚基）的短发夹 RNA 介导的敲低所减弱。蛋白异戊烯化的选择性抑制剂（FTI-277 或 GGTI-2147）显著抑制营养诱导的 ROS 生成，表明一种（或多种）异戊烯化的小 G 蛋白和/或三聚体 G 蛋白的 γ 亚基的激活参与了这个信号轴。用霉酚酸耗尽内源性 GTP 水平，显著降低了这些细胞中葡萄糖诱导的 Rac1 激活和 ROS 生成。其他免疫抑制剂，如环孢菌素 A 或雷帕霉素，不耗尽内源性 GTP 水平，也不能影响葡萄糖诱导的 ROS 生成，表明内源性 GTP 是葡萄糖诱导的 Nox 激活和 ROS 生成所必需的。用百日咳毒素（Ptx）处理 INS 832/13 细胞或大鼠胰岛，Ptx 可以 ADP 核糖基化并抑制抑制性三聚体 G 蛋白（即 G(i)或 G(o)），显著减弱这些细胞中葡萄糖诱导的 ROS 生成，表明在这些信号级联中激活了 Ptx 敏感的 G 蛋白。综上所述，我们的研究结果表明，一种异戊烯化的 Ptx 敏感的信号步骤将 Rac1 的激活与葡萄糖介导的胰腺β细胞中 ROS 生成的信号步骤联系起来。
