Src activation generates reactive oxygen species and impairs metabolism–secretion coupling in diabetic Goto–Kakizaki and ouabain-treated rat pancreatic islets

Abstract

Aims/hypothesis Na + /K + -ATPase inhibition by ouabain suppresses ATP production by generating reactive oxygen species (ROS) and impairs glucose-induced insulin secretion from pancreatic islets. To clarify the signal-transducing function of Na + /K + -ATPase in decreasing ATP production by the generation of ROS in pancreatic islets, the involvement of Src was examined. In addition, the significance of Src activation in diabetic islets was examined. Methods Isolated islets from Wistar rats and diabetic GotoKakizaki (GK) rats (a model for diabetes) were used. ROS was measured by 5-(and 6)-chloromethyl-2′,7′-dichlorofluorescein fluorescence using dispersed islet cells. After lysates were immunoprecipitated by anti-Src antibody, immunoblotting was performed. Results Ouabain caused a rapid Tyr 418 phosphorylation, indicating activation of Src in the presence of high glucose. The specific Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) restored the ouabain-induced decrease in ATP content and the increase in ROS production. Both PP2 and ROS scavenger restored the impaired insulin release and impaired ATP elevation in GK islets, but had no such effect in control islets. PP2 reduced the high glucose-induced increase in ROS generation in GK islet cells but had no effect on that in control islet cells. Moreover, ouabain had no effect on ATP content and ROS production in the presence of high glucose in GK islets. Conclusions/interpretation These results indicate that Src plays a role in the signal-transducing function of Na + /K + -ATPase, in which ROS generation decreases ATP production in control islets. Moreover, ROS generated by Src activation plays an important role in impaired glucoseinduced insulin secretion in GK islets, in which Src is endogenously activated independently of ouabain.

Rapamycin impairs metabolism-secretion coupling in rat pancreatic islets by suppressing carbohydrate metabolism

“…To clarify the link between reduced mitochondrial ATP production in the presence of substrates metabolizable in the Krebs cycle and reduced glucose oxidation by rapamycin, recovery of insulin release and ATP content in the presence of high glucose by ROS scavengers and activity of enzymes in the Krebs cycle were examined. Ouabain-induced endogenous ROS suppresses mitochondrial metabolism in the Krebs cycle, subsequently reducing ATP production, and reduces glucose-induced insulin release and ATP levels in the presence of high glucose, which is recovered by the suppression of endogenous ROS production and by ROS scavenge (Kajikawa et al 2002, Kominato et al 2008). High glucose raises ROS level in b-cells (Bindokas et al 2003, Sakai et al 2003, which is also found in our previous study (Kominato et al 2008).…”

“…Ouabain-induced endogenous ROS suppresses mitochondrial metabolism in the Krebs cycle, subsequently reducing ATP production, and reduces glucose-induced insulin release and ATP levels in the presence of high glucose, which is recovered by the suppression of endogenous ROS production and by ROS scavenge (Kajikawa et al 2002, Kominato et al 2008). High glucose raises ROS level in b-cells (Bindokas et al 2003, Sakai et al 2003, which is also found in our previous study (Kominato et al 2008). However, our previous study shows that ROS scavenging does not affect glucose-induced insulin secretion from control islets, but increases that from GK-diabetic islets.…”

“…ATP contents were determined as previously described (Kominato et al 2008). Briefly, after groups of cultured islets were preincubated at 2 .…”

Metformin suppresses hepatic gluconeogenesis and lowers fasting blood glucose levels through reactive nitrogen species in mice

“…Fluorescence was determined using a spectrofluorophotometer (RF-5300PC; Shimazu, Kyoto, Japan) with excitation wavelength at 502 nm and emission wavelength at 523 nm. After 1 h incubation in the presence or absence of metformin, rotenone, ONOO − or hydrogen peroxide with or without RNS scavenger (5 mmol/l α-tocopherol plus 2.3 mmol/l ascorbate) [27], fluorescence was measured and presented as a ratio with respect to the value at time zero.…”

Mitochondrial protein phosphorylation: instigator or target of lipotoxicity?

“…Because of their fundamental importance to cell homeostasis by governing signal-transduction processes, kinases -especially members of the Rous sarcoma virus oncogene protein kinases (Src) and protein kinase C (PKC) families -are commonly crucial in various pathologies. Intriguingly, homologous to the Src and/or PKC activation was found to govern multiple pathological processes in hyperglycemia [21][22][23] and lipotoxicity [24,25]. Because similar pathways were also described as leading to cellular differentiation [17], the actual physiological outcome might depend on the pattern of activated pathways rather than on a single phenomenon.…”