Role Of Cdc42 In Glutamine/Glutamate Metabolism In Pancreatic β Cells

Background and Objectives: Islet beta cells sense the changes of nutrients in circulating blood and secrete insulin which is the only hormone in the body that lowers blood sugar.Absolute or relative lack of insulin is the basis of diabetes.Cell division cycle protein 42(Cdc42)is a small molecule GTPase belonging to the Rho family.Studies have shown that Cdc42 plays a role in glucose-stimulated insulin secretion,especially for GSIS phase II secretion.Amino acid,one of the three major nutrients,plays a role similar to glucose in the body’s regulation of insulin secretion,and is a physiological stimulating factor that regulates the synthesis and secretion of insulin.More and more studies show that glutamine(GLN)/ glutamic acid(GLU)and its downstream metabolites play an important role in insulin secretion and the development of diabetes.In patients with type 2 diabetes(T2D),plasma glutamine levels are reduced,and glutamine supplementation can improve blood glucose control in patients.Glutaminase(GLS1)is the first key enzyme in the process of glutamine metabolism.It metabolizes glutamine into glutamic acid and enters the tricarboxylic acid cycle to provide energy for cells and provide a substrate for the synthesis of large molecules.Recent studies have shown that Cdc42 can enhance the activity of glutaminase in tumor cells.Whether Cdc42 affects the physiological functions of pancreatic beta cells by regulating glutamine metabolism(eg,metabolic status,energy supply,insulin synthesis and secretion)has not been reported.The purpose of this project is to use the islet β cellspecific knockout of the Cdc42 mouse model and the islet β cell line to clarify the regulatory role and mechanism of Cdc42 on the metabolism of glutamine / glutamic acid and its role in islet β cells.Methods: 1.Mating Cdc42 Lox P mice with pancreatic β-cell-specific Cre(RIP)mice to obtain pancreatic β-cell-specific knockout Cdc42 mice.Genotyping PCR was used to identify whether the RIPCre gene or Lox P flanked Cdc42 gene were exist in mouse genome;Cdc42 F/F(+)with both Cre and Lox P-flanked Cdc42,is a Cdc42 islet β-cellspecific knockout mouse,represented by KO,Cdc42 F/F(-)with only Lox P-flanked Cdc42 as a control mouse,and WT.Isolating mouse islets and performing Western blot/q RT-PCR to analyze the expression level of Cdc42 in mouse islet β cells;2.Using metabolic cages to measure the consuming amounts of diets,drinking water and other metabolic parameters of KO/WT mice with regular tapwater or 20% glucose,respectively;3.After 16 h of starvation treatment,20% glucose solution + PBS(5min)/ 20% glucose + 7.5% glutamine solution(5min)were injected intraperitoneally,respectively,to measure and compare the changes of blood glucose in mice;4.The insulin secretions were measured by Elisa kit in isolated mouse islets and cultured mouse islet β cell line Min6 or human islet β cell line 1.1B4 cell,under Ml141,inhibiting Cdc42 activity or 968 inhibiting glutaminase(GLS1)activity,treatments.5.Western blot/q RT-PCR were used to detect and compare the expression levels of the genes related to glutamine metabolism(GLS1/GDH SLC1A5);6.Cdc42-specific inhibitor ML141,glutaminase-specific inhibitor 968 or Si RNAs were used to detect glutaminase expression,the cell viability,intracellular ATP and glutamate content,and extracellular Oxygen consumption rate(OCR)in Min6 or 1.1B4 cells;7.Western blotting was used to detect the phosphorylation of AMPK protein in pancreatic β cells after above treatments.Results: 1.Islet β-cell conditioned knockout Cdc42 mice were successfully constructed while Cdc42 protein expression and m RNA levels were significantly down-regulated in isolated pancreatic islets.2.There were no significant differences in the amounts of food intake,carbon dioxide production(VCO2),and oxygen consumption(VO2),but the water consumptions in KO group were slightly higher than WT group under normal diets.Water consumption,blood glucose,carbon dioxide production(VCO2),and nighttime oxygen consumption(VO2)were and significantly increased in KO group when supplying 20% glucose H2 O.3.Intraperitoneal injection of 20% glucose solution + PBS(5min)or 20% glucose + 7.5% glutamine solution(5min),the blood glucose of WT mice reaches a peak in ~ 15 min,and the time of peak blood glucose in KO mice is delayed.After 30 minutes of glucose injection,the blood glucose of KO mice was higher than that of WT group,and there was a significant difference.The glucose tolerance of KO mice was obviously impaired.The blood glucose of 20% glucose + 7.5% glutamine(5min)mice in WT group was significantly lower In the 20% glucose solution group,glutamine can alleviate the rise of blood sugar in WT mice.However,the glutamine-lowering effect of glutamine was suppressed in KO mice.4.Glutamine promoteed insulin secretion in the islet β cell line Min6 cells.Glutamine enhances the insulin secretion from isolated mouse islets at basal glucose concentration;meanwhile,it significantly promoted insulin secretion under high glucose conditions.But this effect was significantly inhibited in the absence of Cdc42 in beta cells.5.GLS1 protein and m RNA levels were down-regulated,whereas glutamate dehydrogenase(GDH)and glutamine transporter(SLC1A5)m RNA levels were not significant impacted in β cell specific Cdc42 knockout islets or the inhibited Cdc42 cell lines.6.Glutamine significantly increased the extracellular oxygen consumption,cell activity,and intracellular glutamine content of β cells;inhibiting the expression and activity of GLS1 via si RNA or 968,significantly inhibited cell activity,reduced intracellular ATP contents and glutamate contents;in the meantime,inhibiting Cdc42 activity by its specific inhibitor ML141,significantly reduced extracellular oxygen consumption,cell activity and ATP content induced by glutamine.7.Glutamine had an inhibitory effect on AMPK phosphorylation;Both ML141 and glutaminase specific inhibitor 968 enhanced AMPK phosphorylation in dose dependent manner respectively.Ml141 did not affect the phosphorylation of AMPK upstream kinase LKB1.Conclusion:Islet β-cell specific deletion of Cdc42 inhibits insulin secretion and affects the body’s tolerance to glucose,but has no significant effect on the use of insulin in peripheral tissues.Glutamine can reduce the rise of blood sugar caused by glucose,and has a promoting effect on insulin secretion,but its effect of lowering blood sugar and promoting insulin secretion is suppressed when Cdc42 is absent.Glutamine can increase intracellular glutamate levels,ATP content,enhance cell activity and metabolic capacity.Cdc42 regulates the expression of glutaminase(GLS1),a key enzyme in glutamine metabolism.Moreover,after inhibiting β-cell Cdc42 or GLS1,intracellular glutamate level,ATP content,cell activity,and cell metabolic capacity all decreased synchronously.This indicates that Cdc42 may affect the glutamine metabolism in cells by regulating the expression of glutaminase,and then affect the physiological function of islet β cells.