| Type 2 diabetes mellitus(T2DM)is one of the major chronic diseases worldwide.Nowadays,the number of adult patients with T2 DM in China is nearly 114 million,which ranks first in the whole world.Therefore,the prevention and treatment of diabetes in China is extremely important.The loss and dysfunction of the pancreatic ?-cells is the crucial pathophysiologic event in the onset and development of T2 DM.A major goal of diabetes therapy is to promote the formation of new ?-cells.Pancreatic ductal epithelial cells have been identified as ?-cell precursors,which has attracted high attentions in the field of diabetes in the last decade.In recent years,a significant achievement in the development of novel anti-diabetic drugs is the study and clinical application of GLP-1(glucagon-like peptide 1)analogues as well as DPP-4(dipeptidyl peptidase-4)inhibitors.The physiological effects of GLP-1 are multiple such as promoting insulin secretion,which is depended on GLP-1binding to its receptor GLP-1R to activate downstream signaling pathways subsequently.The potential roles of GLP-1 and its anologues such as Exendin-4(Exenatide)in inducing pancreatic ductal cell differentiation and ?-cell regeneration have been recognized,but the underlying molecular mechanisms are still largely unknown.Puerarin,the main active ingredient of the traditional Chinese herb Radix Puerariae,has been used for the treatment of cardiovascular and cerebrovascular diseases clinically.In our previous study,we identified a novel role of puerarin in protecting ?-cell survival by mechanisms involving activation of GLP-1R signaling and downstream targets.Whether puerarin has the potential effect on promoting the pancreatic ?-cell regeneration is the next question we intend to answer.This study is aimed to investigate the effects of exendin-4 and puerarin on modulating the differentiation of pancreatic ductal epithelial cells into ?-cells mediated by GLP-1R pathway,the underlying mechanismsinvolved need to be assessed as well.HFD(high-fat diet)induced diabetic mouse model and isolated mouse pancreatic ductal cells were used for experiments.The main results are presented as follows.1.Studies in vitro to investigate the effect of Exendin-4 on modulating the pancreatic ductal epithelial cell differentiationOur findings confirmed that Exendin-4,an agonist of GLP-1 receptor(GLP-1R),could induce the differentiation of isolated cultured mouse pancreatic ductal cells significantly.Markers of new ?-cell formation,including positive expressions of insulin,Pdx1,Ngn3,and MafA were all observed in CK19+ pancreatic ductal cells treated by Exendin-4.Also,small islet-like cell clusters(ICCs)were found to originate in the vicinity of the ductal epithelium.However,the insulin secretion ability stimulated by glucose of these new ?-cells was weak.To explore the mechanism related to the differentiation process induced by Exendin-4,GLP-1R antagonist Exendin(9-39),Wnt/?-catenin transcription inhibitor ICG001,and JAK2 inhibitor AG490 were applied.It is revealed Exendin-4 could activate AKT,?-catenin and JAK2/STAT3.Differentiation of pancreatic ductal cells triggered by Exendin-4 was blocked by inhibition of GLP-1R or Wnt signaling strongly,and JAK2/STAT3 signaling acted as a downstream of Wnt pathway.GLP-1R/Wnt/STAT3 pathways work as a cell signal transduction network to regulate pancreatic ductal cell differentiation synergistically.2.Studies in vivo to evaluate the effect of Exendin-4 on promoting the pancreatic ductal epithelial cell differentiation in HFD diabetic miceThe hyperglycemia of HFD diabetic mice were significantly ameliorated by 2 weeks of continuous administration of Exendin-4.Positive expressions of insulin as well as Pdx1 and Ngn3 in CK19+ductal cells were detected in pancreatic sections of HFD mice treated by Exendin-4.These results indicated that in HFD mice,Exendin-4 couldpromote pancreatic ductal cells differentiated into ?-cells,which was consistant with our findings in vitro.3.Studies to evaluate the potential effect of puerarin on the pancreatic ductal epithelial cell differentiation mediated by GLP-1R signalingThe impact of puerarin on pancreatic ductal cell differentiation was invastigated in this project furtherly.The data showed puerarin could stimulate ductal cell differentiation in vitro and in vivo markably by inducing the expressions of insulin and Pdx1 in CK19+ ductal cells.The effect of puerarin was depended on GLP-1R signaling activation,which can be enhanced by Exendin-4,but be blocked by Exendin(9-39).Overall,based on our study of Exendin-4,we clarified that GLP-1R/Wnt/STAT3 pathways work as a cell signal transduction network to regulate pancreatic ductal cell differentiation synergistically.Our findings identified critical targets involved in this process as potential candidates for anti-diabetic drug development.We hope to provide new approaches for diabetes treatment to promote new ?-cells formation.Meanwhile,we evaluated the potential effect of puerarin on promoting pancreatic ductal cell differentiation and new ?-cells formation.These results will contribute to develop strategy for research and application of Chinese antidiabetic herbs. |
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