| Background and ObjectiveType 2 diabetes is characterized by insulin resistance and the dysfunction of pancreaticβcells.The dysfunction ofβcells is early responsible for the impairment of the first phase of glucose-stimulated insulin secretion,but its impaired mechanism is not clear.It is generally accepted that insulin secretion is triggered by a rapid rise of calcium ions concentration due to influx of extracellular Ca2+ and mobilization from intracellular stores. Glucose-stimulated insulin secretion depend on extracellular Ca2+ influx. Insulin granules are connected to the plasma membrane via L-type Ca2+ channel–SNARE (soluble N-ethylmaleimide-sensitive fusion protein -attachment protein receptor) complexs, which benefit the formation of localized high calcium ions concentration and trigger exocytosis of these granules. Although the empty of acidic calcium stores is reported to inhibit insulin secretion(in INS-1cells), now it is not reported that the change of calcium ions content in secretory granules is related to the first phase of insulin secretion. Moreover, at present fluorescent probes are mainly used to indirectly reflect global calcium ions concentration inβcells,and direct localization and quantitative analysis of calcium ions at the subcellsular level are seldomly reported, thus we apply the modified potassium pyroantimonate precipitation method to localize and quantitate calcium ions in secretory granules of rat pancreaticβcells in order to investigate its effect on the first phase of insulin secretion .MethodsMale Sprague–Dawley rats were randomly assigned to one of two dietary groups: high fat diet and normal diet. During the 5 months study, blood samples were taken from the rats by tail bleeding and collected in tubes after an overnight fasting every 7 days, the plasma was frozen and stored at -70°C for later measurement of the plasma insulin. At the end of feeding ,the intravenous glucose tolerance test was done.The plasma level of insulin was detected by using the radioimmunoassay kits.The pancreatic duct of rats was perfused with collagenase V. Islets were partially isolated by Histopaque-1077, hand-picked from the acinar tissue debris. After islets were isolated in the high fat diet and normal diet groups and islets of normal rats were incubated with exogenous insulin for 0 minutes and 240 minutes,the modified potassium pyroantimonate precipitation method was applied to localize and quantitate calcium ions in secretory granules of rat pancreaticβcells.Results1.The fasting plasma level of insulin in Sprague–Dawley rats induced by high fat diet is gradually increased.When it is double or triplication of the basal level of insulin ,the first phase of insulin secretion will be impaired. The result of the electron microscope shows that the content of calcium ions in sub-membraneous secretory granules of pancreaticβcells is less in the high fat diet group than in the normal diet group(P <0.01).2.After normal pancreaticβcells were incubated with exogenous insulin for 240 minutes, the content of calcium ions in sub-membraneous secretory granules is less than that of the control group(P <0.01).The first phase of insulin secretion is severely impaired; IP3 receptor blockers, 2-APB or Xestospongin C ,inhibit the decrease of calcium ions of secretory granules induced by exogenous insulin.The results suggest that insulin could induce calcium ions release from secretory granules through the activation of IP3 receptors on granules.Conclusion1. The high content of calcium ions in insulin secretory granules is crucial for the secretory function of pancreaticβcells2. Insulin could induce calcium ions release from secretory granules through the activation of IP3 receptors on granules, which leads to the impairment of the first phase of insulin secretion. |
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