Role And Mechanism Of CDC42 In Insulin Secretion Of Pancreatic ?-cells

Background and Objectives:Diabetes is caused by imbalance of insulin secretion or insulin resistance of peripheral tissues which leads to absolute or relative deficiency of insulin,resulting in high level of blood glucose and secondary multiple organs injury in the patients who suffer from diabetes.CDC42,a member of Rho family GTPases,has been reported to regulate insulin secretion in vitro.The purpose of this study is to investigate the roles and regulatory mechanisms of CDC42 in insulin secretion of pancreatic b-cells using islet beta cell specific CDC42 knockout mouse model.We tried to define the molecular signaling pathways which might be involved in glucose stimulated insulin secretion(GSIS).This study should provide an insight in elucidating the role and mechanism of CDC42 in insulin secretion of pancreatic beta cells.Methods:1.Analysis of CDC42 expression profile in pancreas and multiple tissues with different age in mice using the bioinformatics database.2.Generation of pancreatic ? cell-specific CDC42 knockout mice,identification of mouse genotype by PCR and confirmation of the CDC42 expression in islets by Western blot.3.Isolation of pancreatic islets and preparation of MIN6 cell model with inhibited CDC42 activity;Measurement of insulin secretion and expression of insulin gene with stimulations of low glucose,high glucose and high KCL.4.The intracellular distributions of CDC42 and insulin protein by immunofluoresence technique with different conditions5.Mechanism analysis of CDC42 regulating insulin secretion: The beta cells were first loaded with calcium ion probe(Fluo-3,AM)and the intracellular calcium concentrations were measured by real-time monitoring of dynamic changes with different conditions.The mRNA expressions of the calcium regulatory proteins(NCX,SERCA,IP3R)were detected by Q-PCR analysis.The protein levels of the related transcriptional factors were confirmed by Western blot analysis for further determining the role and mechanisms of CDC42 mediated intracellular calcium in GSIS.Results:1.CDC42 is highly expressed in the nervous system and the endocrine system in mice and the expression of CDC42 in pancreas was started from the embryo of 14 days,and the expression of CDC42 was significantly increased until reaching to the peak before birth,and then the CDC42 expression was maintained a certain degree from birth to adult.2.Genotyping and Western blot analysis showed that CDC42 expression was significantly down-regulated to 20% in islets of the ?-cell knockout mouse islets compared with control group.3.? cell-specific CDC42 knockout mice down-regulated GSIS,and the insulin secretion was also inhibited in MIN6 cells with inhibited CDC42 activity.4.Inhibition of CDC42 activation in MIN6 resulted in the accumulation of insulin granules,indicating that there is the dysfunction of insulin secretion in ? cells when CDC42 is inhibited.5.The results showed that deletion of CDC42 or inhibiting CDC42 activation in pancreatic ?-cells significantly attenuated the recruitment of the intracellular [Ca2+] in GSIS,and Q-PCR results also showed that the expressions of calmodulin,calcineurin and IP3 R genes were decreased.In addition,the protein level of Foax2 was also down-regulated in the islets of the beta cell specific CDC42 mice.Conclusion:Deletion of CDC42 gene in the islet beta cells or inhibition of CDC42 activity in MIN6 cells led to blockage of insulin granule exocytosis and reduction of insulin secretion,and the mechanism may be related to CDC42 deficiency-induced the lack of intracellular calcium homeostasis.In addition,knockout of CDC42 induced down-regulation of Foxa2 expression may be also involved in insulin secretion in pancreatic beta cell.In summary,our study demonstrates that CDC42 is an important regulator in the glucose stimulated insulin secretion in vivo and in vitro.