Glutamine Role Of Cdc42 In Insulin Synthesis And Secretion Mediated By

Purpose: The development of diabetes mellitus(DM)is associated with an absolute or relative deficiency of insulin,and it is a progressive metabolic disorder characterized by hyperglycemia.Studies have shown that Cdc42 plays a key role in the synthesis and secretion of insulin.In addition,glutamine,a non-essential amino acid,may be involved in the synthesis and secretion of insulin,and its specific mechanism is not clear.We aimed to investigate the role of Cdc42 in glutamine-mediated insulin synthesis and secretion and its molecular mechanism,providing a new strategy for the prevention and treatment of diabetes.Method: 1.Preparation and identification of specific knockout islet β-cell Cdc42 mice Mice with pancreatic β-cell-specific knockout of Cdc42 were obtained by mating Cdc42-Flox mice with RIP-CRE mice(CRE recombinase carrying a rat pancreatic β-cell-specific insulin promoter).Mouse tail tip DNAs were genotyped by PCR.Gold islet cell clusters were manually selected using the collagenase P(CLG)method for Western blot and RT-q PCR experiments to detect Cdc42 protein and m RNA knockdown in islet cells.2.Glucose tolerance test in mice The mice were fasted for 16h(only providing water),then injected with 20% glucose solution(2g/kg)intraperitoneally for 5min,followed by 7.5% glutamine(AlaGLN)solution(0.75g/kg),and the levels of blood glucose were detected at 0min,15 min and 30 min.3.Detection of insulin synthesis and secretion in mice The transcription of INS1,INS2 were detected by RT-q PCR in mouse β-TC6 cells;the expression and secretion of insulin were measured by Western blot and ELISA kit in isolated mouse pancreatic islets.4.The expressions of glutaminase in mouse pancreatic β cells Mouse islet cell line β-TC6 cells were stimulated with different concentrations of glucose or glutamine using Cdc42-specific inhibitor ML141 for 6 h.The expression of intracellular glutaminase(GLS1)and insulin was detected using immunofluorescence;mouse(F/F + and F/F-)pancreatic tissues were isolated in vitro and fixed,dehydrated,embedded,sectioned,and stained for immunohistochemistry to detect the distribution of GLS1 and insulin in islet cells.5.The proteins modulating the expression of glutaminase in pancreatic β cells 1)Mouse islet cell line β-TC6 cells were treated with Cdc42-specific inhibitor ML141 or PCMV6-Cdc42(T17N)-Flag plasmid(dominant inactivated),and mouse(F/F + and F/F-)islets were isolated,and stimulated with different concentrations of glucose or glutamine for the corresponding time,and the expressions of p38 and STAT3 and their phosphorylation were detected by Western blot.2)Mouse β-TC6 cells were treated with glutaminase inhibitor 968,and the expressions of p38 and STAT3 and their phosphorylation were detected by Western blot.Results: 1.Pancreatic β-cell-specific knockout Cdc42 mice were successfully obtained.Cdc42-Flox mice were mated with RIP-CRE mice to obtain pancreatic β-cellspecific knockout of Cdc42 mice.Knockout mice(F/F + or CKO)were with both Lox P and CRE sequences;control mice(F/F-or WT)with only Lox P sequences.The protein and m RNA levels of Cdc42 were significantly lower in the pancreatic islets from Cdc42-CKO mice than those in the control group.2.The role of glutamine in relieving the glucose tolerance were significantly lessened in Cdc42 CKO mice,and the insulin synthesis and secretion mediated by glutamine in synergy with glucose,were significantly reduced in Cdc42-CKO mice.The results of glucose tolerance and ELISA test in mice showed that glutamine significantly improved glucose-mediated glucose tolerance response and significantly increased insulin secretion in mice injected intraperitoneally;however,the blood glucose value of pancreatic β-cell Cdc42-CKO mice was not improved,the insulin content in blood was significantly downregulated,and the insulin content in islet tissue mediated by glutamine and glucose was significantly reduced.3.Inhibition or knockdown of Cdc42 significantly decreased glutaminase and insulin expression in β-cells After inhibition of Cdc42 activity,the expression of glutaminase and insulin in pancreatic β-cells was significantly reduced regardless of low glucose,high glucose,or glutamine stimulation;glutaminase and insulin expression was significantly depressed in pancreatic tissue of pancreatic β-cell-specific knockout Cdc42 mice.4.Inhibition or knockdown of Cdc42 significantly reduces the phosphorylation of P38 and STAT3 in islet β-cells Inhibition of Cdc42 activity significantly reduced PAK1 、 p38,and STAT3 phosphorylation in pancreatic β-cells;islet p38 phosphorylation also showed the same trend after deletion of Cdc42 gene in pancreatic β-cells.5.The phosphorylation of STAT3 were not affected via inhibition of glutaminase activity in pancreatic β-cells Western blot results showed that p38 phosphorylation was significantly decreased after 968 inhibition of GLS1 activity,but STAT3 phosphorylation did not change significantly.Conclusion: Deletion Cdc42 in pancreatic β-cells significantly lessened the role of glutamine in relieving the glucose tolerance,and decreased the synthesis and secretion of insulin which were enhanced by glucose plus glutamine in mice.The expression of glutaminase and insulin in islet β cells with Cdc42 deletion or inactivation was significantly decreased.Cdc42 might modulate GLS1 expression via signal transducer and activator of transcription 3(STAT3)to regulate glutamine metabolism,and the synthesis and secretion of insulin in pancreatic β-cells.