Glucagon-like peptide-1 and gastrin synergistically promote the differentiation of insulin-producing cells / 中华内分泌代谢杂志

Objective@#Glucagon-like peptide-1(GLP-1) and gastrin synergistically promote the differentiation of insulin-producing cells which differentiated from rat bone marrow mesenchymal stem cells (BMSCs).@*Methods@#(1)Prepare IPCs model: pancreatic duodenal homeobox 1 (Pdx-1), neurogenin 3 (Ngn3) combined with V-type tendon fibrosarcoma oncogene homolog A (MafA) co-transfected BMSCs differentiation into IPCs; (2)IPCs were divided into 4 groups: Group A(uninduced group), group B(GLP-1 induction group), group C(gastrin induction group), and group D(GLP-1 combined with gastrin induction group). Cultured in high glucose medium for 7 days, the expression levels of insulin2, Pdx-1, GK, nestin, and glucagon mRNA were detected by RT-PCR. The insulin secretion of each group was detected by ELISA.@*Results@#After cultured for 7 days under high glucose conditions, the morphology of IPCs in each induction group changed significantly, gradually aggregated and formed scattered cell masses, and the combined induction group formed large cell masses. The staining of disulfide brown was reddish brown; The levels of insulin secretion increased gradually on the 0, 3rd, 5th, 7th, and 9th day after induction, and the increase was the most significant in the combined induction group (P<0.05). Compared with group A, the expression of insulin2 and GK in group B and D was significantly up-regulated, the expression of glucagon was down-regulated in group D, the expression of Pdx-1 was down-regulated in group C, and the expression of glucagon was up-regulated (P<0.05). Compared with group B, The expression of insulin2 was down-regulated in group C, and the expression level of glucagon was up-regulated. The expression levels of Pdx-1 and Insulin2 were significantly up-regulated in group D, and the expression level of glucagon was down-regulated (P<0.05). Compared with group C, the expression level of Pdx-1, insulin2 and GK was significantly up-regulated in group D, and the expression level of glucagon was down-regulated (P<0.05).@*Conclusion@#GLP-1 and gastrin synergistically promote the differentiation of IPCs into islet β cells by up-regulating GK and insulin2 and down-regulating glucagon.

Effects of Apelin on glucose toxicity and islet cells PDX-1 expression / 重庆医学

Objective To study the effects of Apelin on glucose toxicity and islet cells PDX-1 protein expression.Methods The islet β cell line NIT-1 cells were incubated in the medium containing different glucose concentrations(normal glucose concentration group 5.6 mmol/L,high glucose concentration group 16.7 mmol/L,extremely high glucose concentration group 33.3 mmol/L) and +/-Apelin-36 respectively for 3 d.Then the basic insulin secretion amount of islet cells and their secretion amount after glucose stimulation were detected.The intracellular insulin content and the PDX-1 protein and mRNA expression were detected.Results Compared with the normal glucose group,the basic insulin secretion,secretion after stimulation and intracellular insulin in the high glucose group and extremely high glucose group were significantly decreased and PDX-1 protein expression was declined(P＜ 0.05);compared with non-adding Apelin group,the basic insulin secretion,secretion after stimulation and intracellular insulin in the adding Apelin high glucose group and extremely high glucose group were significantly decreased and PDX-1 protein expression was decreased(P＜0.05);the insulin level in islet cells of 6 groups was positively correlated with PDX-1 protein expression and had no correlation with PDX-1 mRNA expression.Conclusion Apelin may participate in the glucose toxic effect by decreasing PDX-1 protein expression,causes the decrease of insulin secretion,thus plays a role in the pathogenesis of diabetes.

Effect of GLP-1 on the expression of PDX-1 gene in islet beta cells of type 2 diabetes rats / 实用医学杂志

Objective To observe the effect of liraglutide, an analogue of glucagon like peptide-1 (GLP-1), on the expression of PDX-1 gene in the islet beta cells of type 2 diabetes rats. Methods Sixty male SD rats were divided into 3 groups:normal control group, diabetic model group and GLP-1 group. Normal control group was given normal diet and diabetic model group and GLP-1 group were given high fat diet. After the eighth weekend, the high fat diet group was intraperitoneally injected with streptozotocin (STZ) 30 mg/(kg·bw), and the GLP-1 group was subcutaneously injected with liraglutide 200μg/kg with 2 times per day for 8 weeks. The other groups were treated with normal saline by subcutaneous injection. Real-time fluorescent quantitative reverse transcription polymerase chain reaction (Real-Time RT-PCR) was used to detect the expression of PDX-1 gene in rat pancreatic islet cells. Results The expressions of PDX-1 mRNA in the GLP-1 group and untreated group were significantly lower (P <0.001) than that in the normal control group, but significantly higher than that after GLP-1treatment (P < 0.001). Conclusion GLP-1 can upregulate the expression of PDX-1 gene in the islet beta cells of type 2 diabetes rats.

Inflnence of benzyl propionate nandrolone in Nampt expression in NIT-1 cells and insulin secretion / 吉林大学学报(医学版)

Objective To study the effects of benzyl propionate nandrolone (BPN ) on the nicotinamide phosphoribosyl transferase (Nampt), insulin receptor substeate-2 (IRS-2 )and pancreatic duodenal homeobox-1 (PDX-1)expressions, cell cycle changes as well as insulin secretion in pancreatic islet cell NIT-1 lines, and to explore the influence of BPN in the Nampte xpression in NIT-1 cells and insulin signaling molecules in high glucose oxidation stress.Methods The NIT-1 cells were cultured with different concentrations (5.6,11.1,16.7,and 27.6 mmol·L-1)of glucose,then they were treated with 10 mg·L-1 BPN for 48 h with no BPN treatment as corresponding control groups.The expression levels of Nampt,IRS-2,and PDX-1 were tested by Western blotting assay.The changes of cell cycle were determined by FCM and the cell insulin secretion levels were measured with radioimmunoassay.Results Compared with corresponding control groups,the expression levels of Nampt,IRS-2, and PDX-1 proteins in the NIT-1 cells in various BPM groups were increased (P<0.05 or P<0.01).The G0/G1 phase arrest was relieved (P<0.01)when the cells was cultured in low glucose (5.6 mmol·L-1 )condition,and the G2/M block was remitted significantly in high glucose (27.6 mmol·L-1 )condition (P<0.01),furthermore, the cell insulin secretion was promoted compared with control groups except 1 1.1 mmol· L-1 glucose group (P<0.01).Conclusion BPN can promote the expression levels of Nampt,ISR-2 and PDX-1 proteins in NIT-1 cells. There is close relationship between the Nampt expression in NIT-1 cells and insulin signaling pathway and BPN prevents the cells from insulin resistance.

B-cell translocation gene 2 positively regulates GLP-1-stimulated insulin secretion via induction of PDX-1 in pancreatic beta-cells

Glucagon-like peptide-1 (GLP-1) is a potent glucoincretin hormone and an important agent for the treatment of type 2 diabetes. Here we demonstrate that B-cell translocation gene 2 (BTG2) is a crucial regulator in GLP-1-induced insulin gene expression and insulin secretion via upregulation of pancreatic duodenal homeobox-1 (PDX-1) in pancreatic beta-cells. GLP-1 treatment significantly increased BTG2, PDX-1 and insulin gene expression in pancreatic beta-cells. Notably, adenovirus-mediated overexpression of BTG2 significantly elevated insulin secretion, as well as insulin and PDX-1 gene expression. Physical interaction studies showed that BTG2 is associated with increased PDX-1 occupancy on the insulin gene promoter via a direct interaction with PDX-1. Exendin-4 (Ex-4), a GLP-1 agonist, and GLP-1 in pancreatic beta-cells increased insulin secretion through the BTG2-PDX-1-insulin pathway, which was blocked by endogenous BTG2 knockdown using a BTG2 small interfering RNA knockdown system. Finally, we revealed that Ex-4 and GLP-1 significantly elevated insulin secretion via upregulation of the BTG2-PDX-1 axis in pancreatic islets, and this phenomenon was abolished by endogenous BTG2 knockdown. Collectively, our current study provides a novel molecular mechanism by which GLP-1 positively regulates insulin gene expression via BTG2, suggesting that BTG2 has a key function in insulin secretion in pancreatic beta-cells.

NKX6.1 combined with PDX1 induces mesenchymal stem cell differentiation into B-like cells / 第二军医大学学报

Objective To study the synergistic effect of NKX6. 1 and PDX1 in inducing differentiation of fetal liver-derived mesenchymal stem cells(FL-MSCs) into the pancreatic B cells and to explore the underlying mechanisms, so as to obtain enough islet-like body for transplantation. Methods Recombinant adenovirus vector harboring both PDX1 and NKX6. 1 genes was constructed, and the vector was used to infect FL-MSCs. Then a series of cytokines were used to induce the differentiation of infected FL-MSCs into pancreatic B cells. The expressions of PDX1, NKX6.1 gene, transcription factors NGN3, NeuroDl/Beta2, MafA as well as C-peptide were examined. Results PDX1 and NKX6. 1 were detected in FL-MSCs cells 24 h after infection; cells began to express NGN3, NeuroDl, and MafA and stably expressed pancreatic B cell related factors including insulin after induction. The expression of these molecules was in a certain order. Conclusion PDX1, NKX6. 1 combined with a series of cytokines can effectively induce FL-MSCs to differentiate into pancreatic islet B cells in vitro, which might be through activation of transcription factors NGN3, NeuroDl, and MafA in turn, inducing FL-MSCs to differentiate towards endocrine precursor cells, B endocrine precursor cells and B cells in turn.

Construction and expression of human PDX-1 eukaryotic expression vector / 上海第二医科大学学报

Objective To construct eukaryotic expression vector of human pancreatic duodenal homeobox 1(PDX-1) gene,and to detect its expression in NIH3T3 cell lines. Methods The whole coding sequence of PDX-1 gene was amplified by polymerase chain reaction(PCR) from human pancreatic-cell tumors cDNA.The fragment was inserted into eukaryotic expression vector pcDNA3.1 plasmid.The recombinant plasmid was verified by double digestion and DNA sequencing.The expression of PDX-1 gene in NIH3T3 cells was assayed by Western blot. Results The length of specific fragment amplified by PCR was 852 bp,and the recombinant plasmid pcDNA3.1-PDX-1 showed two bands of 5.5 kb and 852 bp by digestion using respective restriction enzymes BamHⅠand EcoRⅠ.The sequence of PDX-1 gene was approved or confirmed by blasting to GenBank.It was suggested that PDX-1 gene had been cloned into pcDNA3.1 vector correctly.Western blot showed that PDX-1 gene was expressed,which was detected 24 h after pcDNA3.1-PDX-1 plasmid was transfected into NIH3T3 cells. Conclusion The recombinant eukaryotic expression vector pcDNA3.1-PDX-1 was successfully constructed and expressed in NIH3T3 cell lines.

Pancreatic duodenal homeobox-1 advances transdiffenentiation of bone marrow mesenchymal stem cells into insulin-producing cells in vitro / 医学研究生学报

Objective: To investigate the possible effects of Pancreatic duodenal homeobox-1((Pdx-1))expression in transdifferentiation of bone marrow mesenchymal stem cells(MSC) in vitro. Methods: The eukaryotic expression vector containing Pdx-1 was constructed.After such vector was transfected into MSC using Superfect,G418 was used to select the positive cells.Then both(Pdx-1~(+)MSC)and((Pdx-1~-MSC)) were induced to transdifferentiate in vitro.The expressions of insulin were analyzed by immunohistochemistry staining. Results: Restricted enzyme analysis and sequencing showed that interest gene segment was consistent with that in Gen Bank,Recombination vector was effectively transformed into MSC demonstrated by fluorescence microscope;insulin-producing cells from Pdx-1~(+)MSC were higher than that from Pdx-1~(-)MSC[(28.23?2.56)% and(7.08?2.69)%,respectively]. Conclusion:Pdx-1 can promote adult rat MSC to transdifferentiate into insulin-producing cells in vitro,and this approach might lead to a widespread cell replacement therapy for type I diabetes.

Construction of pancreatic duodenal homeobox-1 recombinant retroviral vector and its stable expression in liver progenitor cells / 第二军医大学学报

Objective:To construct a recombinant retroviral vector expressing pancreatic duodenal homeobox-1(PDX-1) gene and express the PDX-1 expression cassette in liver progenitor cells(LEPCs),so as to study the conversion of hepatic cells to pancreatic-like cells.Methods: The full length of PDX-1 gene was amplified by PCR and subcloned into pMSCVpuro vector.The pMSCV PDX-1 puro was then introduced into Phoenix package cells and the cell culture supernatant was used for Ping-Pong infection of another packaging cell line PT67 to obtain stable virus-producing cell line.Results: The pMSCV PDX-1 puro vector was successfully constructed and transfected into PT67 cells.The viral supernatants of PT67 cells efficiently infected LEPCs in vitro and the infected LEPCs stably expressed PDX-1 gene.Conclusion: The pMSCV PDX-1 puro vector and PDX-1 expressing LEPCs have been successfully constructed,laying a basis for studying the transdifferentiation of liver progenitor cells to pancreas lineage cells.

High expression of PDX-1 in bone marrow mesenchymal stem cells mediated by superfect / 中国病理生理杂志

AIM:To construct a eukaryotic expression vector containing pancreatic duodenal homebox-1(PDX-1)and to elevate the expression efficiency of exogenous gene in rat bone marrow mesenchymal stem cells(MSCs).METHODS:Recombinant vector containing PDX-1 was constructed.Flow cytometry was used to identify the cell cycle of bone marrow mesenchymal stem cells(MSCs)cultured in vitro.Recombinant vector containing PDX-1 was transfected into bone marrow MSCs using superfect in medium.After being selected by G418,RT-PCR and Western blotting were used to investigate the expression of PDX-1 in MSCs.RESULTS:Restricted enzyme analysis and sequencing showed that PDX-1 gene segment was consistent with that in GenBank.Flow cytometry showed that there were about 85.9% cells at the cell cycle of G_0/G_1.The whole cells transfected emitted green fluorescence under flow cytometry.The efficiency of transfection was above 40%.RT-PCR and Western blotting demonstrated that there was expression of PDX-1 in transfected bone marrow MSCs.CONCLUSION:Recombinant vector containing PDX-1 was constructed successfully.Superfect mediated expression of exogenous gene in bone marrow MSCs in a high efficiency,and bone marrow MSCs containing exogenous gene are an ideal cells for gene therapy.