The Study Of Amylin On The Biological Effects Of Pancreatic Islet Cells From Neonatal SD Rat

Aims :Amylin is the main component of pancreatic amyloid deposits in vast majority of patients and animals with type 2 diabetes . It is a normal secretory product of the pancreatic -cells. Clinical pathology found that the degree of islet (prodominantly 13 -cells) mass replaced by amyloid might be a determination of the severity of the diabetic disease process . These demonstrated that amylin took a important part in the progress and development of type 2 diabetes. Until now the stttdy of amylin on the biological functions is still not clear , especially little known about the changes of ultrastructure effects to insulin secretion . Using neonatal SD rat pancreatic islet cells in vitro the present study was to investigate the mechanisms of amylin on the biological effects , by determining insulin secretion , changes of DNA and insulin contents in cells , intracellular free calcium concentration ([Ca2]1) , membrane potential (MP) , p11 , reactive oxygen species (ROS) and mitochondria activity. Methods 1. Isolated pancreatic islet cells from neonatal SD rat were cultured in monolayer in vitro. Using radioimmunoassay , ultraviolent spectrophotometer we measured insulin secretion , changes of DNA and insulin contents in cells in different concentrations of amylin incubated 2. Fluctuations of intracellular pH , RQS in 13 -cells were detected by using adherent cell analysis sorting (ACAS57O) and MTT assay for mitochondria activity aftre 10 ii rnol/L amylin incubated for 2 hours . 3. With the molecular fluorescent probes and digital imaging techniques of advanced of laser confocal microscope , we monitored the fluctuations of [Ca2, MP in single 13 -cells exposed to different concentrations of amylin accurately . We also investigated the mechanisms of changes in [Ca2] by 16.7 mmol/L glucose stimulation , seting tip calcium channel blocker (verapamil) group and inhibitor of cGMP cyclase (methylene blue) group preincubated by 10 ii mol/L amylin Results I. The basal insulin secretion was not significantly affected after arnylin incubated for 2 hours . I-lighter concentrations of arnylin could dose-depend inhibit insulin secretion at 16.7 mmol/L glucose stimulation (I <0.01) , no significant differences in response to lower concentrations of amylin . Changes of DNA and insulin contents in cells exposed to highter concentrations of amylin rose significantly compared with controls (P< 0.01) . Whereas lower concentrations of amylin had no differences . 2. 10 i? mol/L amylin caused an elevation of [pH], an increase of intracellular ROS contents and a remarkable decrease of cell activity at the beginning of 1 .5 hours incubated . Morphological observation found that portions of cells appeared cell body shrinking , kytoplasm vacudus degenrating and few occurred nucleus pycnosis , cracking , umbo of cytomembrane by light microcope . 3. Exposed to highter concentrations arnylin for 2 hours , f3 - cells showed a significant rise of fluctuations of [Ca2 and fluctuations of MP decrease compared with controls (P <0.05) . The results suggested highter concentrations of amylin rise [Ca2 and cause membrane hyperpolarization . But the exprosure of pancreatic P -cells to highter concentrations of amylin dose-depend inhibited an ability of glucose to provoke [Ca2] rise and caused continuous membrane hyperpolarization Changes of [Ca2] , MP did not differ significantly in lower concentrations of amylin group compared with control group (P>0. 05) . In the study , we also foun...