Control of Insulin Secretion by Production of Reactive Oxygen Species: Study Performed in Pancreatic Islets from Fed and 48-Hour Fasted Wistar Rats

Mitochondria and NADPH oxidase are important sources of reactive oxygen species in particular the superoxide radical (ROS) in pancreatic islets. These molecules derived from molecular oxygen are involved in pancreatic β-cells signaling and control of insulin secretion. We examined the involvement of ROS produced through NADPH oxidase in the leucine- and/or glucose-induced insulin secretion by pancreatic islets from fed or 48-hour fasted rats. Glucose-stimulated insulin secretion (GSIS) in isolated islets was evaluated at low (2.8 mM) or high (16.7 mM) glucose concentrations in the presence or absence of leucine (20 mM) and/or NADPH oxidase inhibitors (VAS2870–20 μM or diphenylene iodonium—DPI—5 μM). ROS production was determined in islets treated with dihydroethidium (DHE) or MitoSOX Red reagent for 20 min and dispersed for fluorescence measurement by flow cytometry. NADPH content variation was examined in INS-1E cells (an insulin secreting cell line) after incubation in the presence of glucose (2.8 or 16.7 mM) and leucine (20 mM). At 2.8 mM glucose, VAS2870 and DPI reduced net ROS production (by 30%) and increased GSIS (by 70%) in a negative correlation manner (r = -0.93). At 16.7 mM glucose or 20 mM leucine, both NADPH oxidase inhibitors did not alter insulin secretion neither net ROS production. Pentose phosphate pathway inhibition by treatment with DHEA (75 μM) at low glucose led to an increase in net ROS production in pancreatic islets from fed rats (by 40%) and induced a marked increase (by 144%) in islets from 48-hour fasted rats. The NADPH/NADP+ ratio was increased when INS-1E cells were exposed to high glucose (by 4.3-fold) or leucine (by 3-fold). In conclusion, increased ROS production through NADPH oxidase prevents the occurrence of hypoglycemia in fasting conditions, however, in the presence of high glucose or high leucine levels, the increased production of NADPH and the consequent enhancement of the activity of the antioxidant defenses mitigate the excess of ROS production and allow the secretory process of insulin to take place.

线粒体与烟酰胺腺嘌呤二核苷酸磷酸氧化酶（NADPH oxidase）是胰岛内活性氧簇（reactive oxygen species, ROS，尤其是超氧阴离子自由基）的重要来源。这类由分子氧衍生而来的活性氧分子参与胰腺β细胞的信号转导，并调控胰岛素的分泌过程。本研究针对进食组与禁食48小时的大鼠胰岛，探讨了NADPH氧化酶来源的ROS在亮氨酸及/或葡萄糖诱导的胰岛素分泌中的作用。本研究在低浓度（2.8 mM）与高浓度（16.7 mM）葡萄糖条件下，分别设置添加或不添加亮氨酸（20 mM）及/或NADPH氧化酶抑制剂（VAS2870：20 μM，或二苯基碘鎓盐DPI：5 μM）的组别，对分离得到的胰岛的葡萄糖刺激胰岛素分泌（glucose-stimulated insulin secretion, GSIS）水平进行评估。采用二氢乙锭（dihydroethidium, DHE）或MitoSOX Red荧光染料对胰岛进行20分钟染色后，通过流式细胞术检测细胞分散后的荧光强度，以此定量ROS的生成水平。本研究还在培养的INS-1E细胞（一种胰岛素分泌细胞系）中，检测了分别添加葡萄糖（2.8 mM或16.7 mM）与亮氨酸（20 mM）后，细胞内NADPH的含量变化。在2.8 mM葡萄糖浓度条件下，VAS2870与DPI可分别使净ROS生成量降低30%，同时使GSIS水平升高70%，二者呈显著负相关（相关系数r=-0.93）。而在16.7 mM葡萄糖或20 mM亮氨酸单独处理的条件下，两种NADPH氧化酶抑制剂均未对胰岛素分泌与净ROS生成量产生显著影响。在低葡萄糖条件下，使用脱氢表雄酮（DHEA，75 μM）抑制戊糖磷酸途径后，进食组大鼠的胰岛净ROS生成量升高40%，而禁食48小时大鼠的胰岛净ROS生成量则显著升高144%。当INS-1E细胞暴露于高浓度葡萄糖（升高4.3倍）或亮氨酸（升高3倍）环境中时，细胞内NADPH/NADP+比值均显著升高。综上，NADPH氧化酶介导的ROS生成增加可在禁食状态下预防低血糖的发生；然而在高葡萄糖或高亮氨酸水平的条件下，NADPH生成量的增加及其后续引发的抗氧化防御系统激活可缓解过量ROS的产生，保障胰岛素分泌过程的正常进行。