A New Way For Endogenous Formation And Growth Of Beta Cells:the Mechanism Of Transdifferentiation From A Cells To P Cells Induced By Glucagon-like Peptide-1(GLP-1)

[Background]The prevalence of diabetes,both type 1(T1D)and type 2(T2D),is increasing throughout the world at an alarming rate.Deficiency of β-cell mass and failure of islet function are major characteristics in both type 1 and type 2 diabetes.The treatment of diabetes has involved both pharmacologic and cellular approaches.Cellular approaches include pancreas transplants and the transplantation of donor islets into the livers of T1D patients.Although helpful,these exogenous treatments currently in use for the treatment of diabetes are not fully effective in their normalization of glucose homeostasis.Therefore,endogenous P-cell mass restoration and islet function reconstruction hold great promise for diabetic therapy.Formation of new beta cells is believed to be possible by the stimulation of beta cell neogenesis from stem/progenitor cells that exist in the pancreatic ducts,or by the differentiation of exocrine stem-like cells into beta cells.A recent discovery has been the finding that alpha cells,specifically immature alpha cells,pro-alpha cells,are precursors of beta cells.Numerous studies have showed that infusion of GLP-1 can efficiently ameliorate hyperglycemia in diabetic models and restore the β-cell mass;however,the mechanism involved is largely unknown.Some researches believe that GLP-1 activates GLP-1 receptor,enhances the reactivity of PDX-1 through PI3K/Akt/FOXO1 pathway.PDX1,a master regulator of the beta cell phenotype,not only activates genes essential for beta cell identity but also represses those associated with alpha cell identity.We hypothesized that the augmentation ofβ-cell mass induced by GLP-1 infusion may,at least in part,trans-differentiation of a cells within the pancreas by possibly PI3K/Akt/FOXO1 pathway through GLP-1 receptor.[objectives]In this study,we will establish a single large dose of STZ-induced diabetes model.We will investigate whether GLP-1 can promote the regeneration of β cells in rat pancreatic islets and its possible mechanism by observing the changes of endocrine cells in rat pancreatic islets after GLP-1 intervention.After application of GLP-1 receptor inhibitors and PI3K enzyme inhibitors,we explore the role of GLP-1 in the process of transdifferentiation from a cells into β cells by measuring the transcription factor and protein expression of PI3K/Akt/FOXO1 signal pathway.[content]The whole project includes three parts:Part I GLP-1 induces neogenesis of β cells in diabetic animal model induced by single high dose of STZ.ObjectivesTo investgate weather GLP-1 can induce the neogenesis of β cells.MethodThe diabetic animal model was induced by single high dose of STZ(60mg/kg,i.p.)injection.Only mice with a blood glucose greater than 28 mmol/L(checked 72 hours after alloxan injection)were selected for experiments.Then the rats were divided into five groups:normal group,diabetic group,GLP-1 50ug/kg group,GLP-1 100ug/kg group,GLP-1 200ug/kg group.The blood glucose was detected and the weight was measured every week.When the intervention was over,ELISA measured surum insulin and C-peptide of the rats,and the number of endocrine cells was tested by insulin/glucogan immunofluorescence.We considered p-values below 0.05 as statistically significant.For all statistical analysis,spss 17.0 was used.All results are expressed as mean±SD.ResultFollowing STZ injection,greater than 90%of p-cells were eliminated.Compared with diabetic group,the weight and the blood glucose of the GLP-1 group decreased in a dose dependent manner.However,even treated with the maximum dose of 200 ug/kg GLP-1,the blood glucose of diabetic rats stayed high level(>19mmol/L).Treatment with GLP-1 induced the increase of serum insulin and C-peptide.The number of insulin express cells increased in a dose dependent manner.ConclusionThe infusion of GLP-1 resulted in neogenesis of β cells.Part Ⅱ GLP-1 promotes endogenous β-cell regeneration and functional reconstruction by driving a-cell transdifferentiation into β cellsObjectivesTo explore the possible mechanism of the regeneration of β cells induced by GLP-1.MethodThe group were divided as part 1.The pancreatic pancreas frozen sections were stained with insulin/glucagon,insulin/amylase,insulin/Pan-CK double immunofluorescence staining,and pancreatic paraffin sections were immunohistochemically stained with PCNA.The statistical method is one-way ANOVA.ResultAfter GLP-1 intervention,insulin/glucagon double positive cells appeared in the islets of rats,and increased in a GLP-1 dose dependent manner.The difference was statistically significant(P<0.05).No insulin/amylase,insulin/pan-ck double positive cells were observed in pancreatic sections.There was no significant difference in PCNA immunohistochemistry(P>0.05),and the staining area was located in the area of α cell distribution.ConclusionGLP-1 promotes the regeneration of β cells from the transdifferentiation of a cells.PartⅢ GLP-1 and its receptors up-regulate PDX-1 via PI3K/Akt/FOXO1 pathway to promote transdifferentiation from a cells into 3 cells.ObjectivesTo investgate weather GLP-1 enhances the expression of transcription factor PDX-1 and MafB and down-regulates the expression of MafA through PI3K/Akt/FOXO1 pathway,then promotes the transdifferentiation from α cells into β cells.MethodThe rats were divided into 7 groups:normal control group,diabetic group,GLP-1 50ug/kg group,GLP-1 100ug/kg group,GLP-1 200ug/kg group,GLP-1 and GLP-1 receptor antagonist exendin(9-39)co-intervention group,GLP-1 and PI3K enzyme inhibitor LY294002 co-intervention group.The expression of GLP-1 R,AKT,PDX-1,MafA,MafB and Foxo1 mRNA was detected by RT-PCR.Western blot was used to test the expression of GLP-1R,PDX-1,MafA,MafB and Akt in rat pancreatic islets.ResultThe expression of GLP-1 R,Akt,PDX-1,Foxo1 and MafA,MafB in the diabetic model group was significantly lower than that in the normal group.The expression of GLP-1 R,Akt,PDX-1,Foxol,MafA mRNA increased significantly after injection of GLP-1,and the expression of MafB decreased in a dose-dependent manner.The expression of GLP-1R,Akt,PDX-1 and Foxol MafA mRNA was decreased compared with GLP-1 100ug/kg group,while the expression of MafB was increased after exendin(9-39)inhibited GLP-1 receptor.The expression of GLP-1 R,AKT,PDX-1,Foxol and MafA mRNA decreased compared with GLP-1100ug/kg,while the expression of MafB increased after LY294002 inhibited PI3K enzyme.The results of western blot were similar to that of PCR.The differences were statistically significant(P<0.05).ConclusionGLP-1 enhances the expression of transcription factor PDX-1 and MafB and down-regulates the expression of MafA through PI3K/Akt/FOXO1 pathway,then promotes the transdifferentiation from α cells into β cells.