thapsigargin and 6-hydroxy-2-5-7-8-tetramethylchroman-2-carboxylic-acid

Insulin secretion involves complex events in which the mitochondria play a pivotal role in the generation of signals that couple glucose detection to insulin secretion. Studies on the mitochondrial generation of reactive oxygen species (ROS) generally focus on chronic nutrient exposure. Here, we investigate whether transient mitochondrial ROS production linked to glucose-induced increased respiration might act as a signal for monitoring insulin secretion.. ROS production in response to glucose was investigated in freshly isolated rat islets. ROS effects were studied using a pharmacological approach and calcium imaging.. Transient glucose increase from 5.5 to 16.7 mmol/l stimulated ROS generation, which was reversed by antioxidants. Insulin secretion was dose dependently blunted by antioxidants and highly correlated with ROS levels. The incapacity of beta-cells to secrete insulin in response to glucose with antioxidants was associated with a decrease in ROS production and in contrast to the maintenance of high levels of ATP and NADH. Then, we investigated the mitochondrial origin of ROS (mROS) as the triggering signal. Insulin release was mimicked by the mitochondrial-complex blockers, antimycin and rotenone, that generate mROS. The adding of antioxidants to mitochondrial blockers or to glucose was used to lower mROS reversed insulin secretion. Finally, calcium imaging on perifused islets using glucose stimulation or mitochondrial blockers revealed that calcium mobilization was completely reversed using the antioxidant trolox and that it was of extracellular origin. No toxic effects were present using these pharmacological approaches.. Altogether, these complementary results demonstrate that mROS production is a necessary stimulus for glucose-induced insulin secretion.

Topics: Adenosine Triphosphate; Animals; Calcium; Chromans; Glucose; Insulin; Insulin Secretion; Islets of Langerhans; Kinetics; Male; Mitochondria; NAD; Rats; Rats, Wistar; Reactive Oxygen Species; Signal Transduction; Superoxide Dismutase; Superoxides; Thapsigargin

Cytosolic Ca2+ mobilization, especially Ca2+ entry, is enhanced in platelets from type 2 diabetic individuals, which might result in platelet hyperaggregability. In the present study, we report an increased oxidant production in resting and stimulated platelets from diabetic donors. Pretreatment of platelets with catalase or trolox, an analog of vitamin E, reversed the enhanced Ca2+ entry, evoked by thapsigargin plus ionomycin or thrombin, observed in platelets from diabetic subjects, so that in the presence of these scavengers Ca2+ entry was similar in platelets from healthy and diabetic subjects. In contrast, mannitol was without effect on Ca2+ mobilization. Catalase and trolox reduced thrombin-induced aggregation in platelets from type 2 diabetic subjects, while mannitol did not modify thrombin-induced platelet hyperaggregability. We conclude that H2O2 and ONOO- are likely involved in the enhanced Ca2+ mobilization observed in platelets from type 2 diabetic patients, which might lead to platelet hyperactivity and hyperaggregability.

Topics: Calcium; Case-Control Studies; Catalase; Chromans; Diabetes Mellitus, Type 2; Humans; Hydrogen Peroxide; Ionomycin; Mannitol; Peroxynitrous Acid; Platelet Aggregation; Reactive Oxygen Species; Thapsigargin