| Background: Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia.Effective treatment of diabetes mellitus is still a medical problem.Islet can secrete a variety of hormones and is the main organ of regulating blood glucose homeostasis.The pancreatic islet is composed of five types of endocrine cells,including β cells,α cells,δ cells,PP cells and ε cells.β cells synthesize and secrete insulin,which reduces blood sugar by promoting glucose uptake and the synthesis of glycogen and lipid in peripheral cells.Diabetic patients often show a decrease in the number of islets,reduced insulin synthesis and secretion of islet βcells,loss of number and quality of islet β cells.Because the integrity of the structure and function of islet β cells is crucial for maintaining blood glucose homeostasis,major studies are exploring the factors and underlying mechanism for β-cell homeostasis.Transcription factors play an important role in the differentiation,maturation and characteristic maintenance of pancreatic islet endocrine cells.Epithelial cells transiently expressing transcription factor Neurogenin3(NGN3)are called endocrine progenitor cells,which differentiate into α,β,δ,PP and ε cell under the regulation of specific transcription factors,such as PDX1,MAFA,HLXB9,PAX6,ISL1,NKX6.1,FOXA2,PAX6,RFX,MAFB,Arx,PAX4 and NKX2.2 These cells constitute pancreatic islets.In the differentiation of endocrine progenitor cells into α and β cells,transcription factors ARX and PAX4 antagonize each other.Progenitor cells expressing ARX and PAX4 differentiate into α cells and β/δ cells,respectively.Maf B is restricted in the α cells to promote the maturation and maintenance of cell properties.Ma FA is restricted in the β cells to regulate the transcription of insulin and GLUT2,which together with PDX1,NKX6.1 and Neuro D,promote the cell integrity.Nuclear factor Y(NF-Y)is a transcription factor widely expressed in cells.It binds to the CCAAT element in the gene promoter region,so it is also called CCAAT binding factor(CBF).NF-Y is composed of three subunits including NFYA(CBF-B),NF-YB(CBF-A)and NF-YC(CBF-C),which form a polymer and bind to CCAAT elements.NF-Y is involved in the regulation of endoplasmic reticulum stress,cholesterol fatty acid metabolism,hepatic gluconeogenesis,cell cycle and many other physiological functions.NF-Y dysfunction is associated with metabolic syndrome,neurodegenerative diseases,hematopoietic stem cell dysfunction and other diseases.NF-Y also regulates cell proliferation by binding the promoters of Cyclin B1,CDK1,CDC25 C,CDC25B and Topoisomerase IIA.NF-Y inactivity leads to cell cycle stagnation in the G2/M phase.NF-Y is involved into the glucose and lipid metabolism,mice with Nf-ya specifically defected in adipocytes showed severe lipid metabolism disorder and fat atrophy.Mice with hepatocellular specific deletion of Nf-ya showed reduced gluconeogenesis.NF-Y and FOXA2 can promote GLUT2 expression by binding to its promoter,and thereby increase fasting blood glucose.Genetics variants of Kcnq1 gene encoding potassium ion channel Kv7.1α subunit were significantly associated with the occurrence of type II diabetes mellitus.Kcnq1 gene variants affect the binding of NF-Y to gene promoter and thereby the gene expression.Moreover,Kcnq gene is physically linked to genes encoding cyclic-dependent protein kinase inhibitor 1C(CDKN1c)and insulin secretion regulator TRPM5 on chromosome,implying that NF-Y may be involved in regulation of β-cell function and development.In conclusion,NF-Y plays an important role in regulating insulin target organs(liver and fat)to maintain glucose homeostasis.Islet β cells have fundamental effects on blood glucose levels in vivo,NF-Y may be involved in the regulation of the proliferation,maturation,insulin synthesis and secretion of pancreatic islet β cells,which remain unclear.Elucidation of the effect of NF-Y on islet function and blood glucose homeostasis is helpful to understand the islet proliferation and development,insulin secretion and β-cell maturation.This will provide new target for diabetes therapy.Purpose: In this study,we studied the effects of NF-Y on the β-cell maintenance,insulin secretion and β-cell maturation of pancreatic islets,and elucidated the related mechanisms.Method:1.The changes of NF-YA expression in islets were observed in high fat diet(HFD),STZ-induced diabetes model,obese mice(OB/OB mice)and old mice.2.In mice with a Nf-ya knockout specially in islets β-cell(Nf-ya βKO),we examined the glucose homeostasis and islet morphology of mice,proliferation and apoptosis of β-cells,development and maturity of β-cells,and the changes in insulin secretion of islet β-cells under glucose stimulation in vitro and in vivo.3.The effects of NF-Y deletion or overexpression on the proliferation,glucose absorption,mitochondrial function and insulin secretion of pancreatic islet β cells were investigated by in vitro and in vivo experiments.4.The integrity of insulin secretion signaling pathway in β cells was detected by dynamic changes of calcium ion fluorescence.Cell proliferation markers,β-cell maturation markers and cytoskeleton system were measured by immunofluorescence staining.Glucose analogue uptake assay and luciferin reporter gene assay were conducted to investigate the effect of NF-Y deletion on glucose uptake and GLUT2 expression in islets.Mitochondrial function was evaluated by glucose changes.The mitochondrial structure and the expression of proteins related to function were detected by transmission electron microscopy,fluorescence quantitative PCR or IHC.Proteome expression profile was used to analyze the regulation of NF-Y on β-cell function.Results:1.NF-YA levels were correlated with the diabetes-related phenotypes.NF-Y expression was significantly decreased in pancreatic islets of STZ treated mice,but was significantly increased in pancreatic islets from HFD-fed mice,Ob/Ob mice and aged mice.2.Under standard chow diet,Nf-ya βKO mice showed relatively higher blood glucose level and lower insulin concentration,while they had similar weight gain and food intake to control mice.Although no difference in insulin tolerance(ITT),Nf-ya βKO mice had impaired glucose tolerance and glucose-stimulated insulin secretion(GSIS),which were further amplified in mice given high fat diet.3.Nf-ya βKO mice had decreased islets in size,reduced mass and quantity of βcells,and decreased amount of pancreatic insulin.The transcription and translation levels of insulin gene INS1/INS2 and insulin transcription regulation gene MAFA/PDX1/NEUROD1 in islets of Nf-ya βKO mice were significantly decreased compared to the control mie.The proportion of alpha cells in the islets increased,suggesting that the structure of the islets was changed.The expression of β-cell proliferation related protein Ki67/PCNA/Cyclin D1 was significantly reduced,and the proliferation ability of β-cell lines was decreased.Overexpression of NF-Y in vitro enhanced the proliferation ability of β-cell lines.There was no change in islet apoptosis in Nf-ya βKO mice.Proteomic analysis showed that the levels of proteins functioning in cytoskeleton were reduced in Nfya βKO islets.4.The expressions of transcription factors MAFA and PDX1 and glucose transporter GLUT2,which are also important β-cell maturity markers,were significantly decreased in Nf-ya βKO islets from mice at postnatal 14 days(P14).Nf-ya βKO mice showed hyperglycemia at P14.5.The islet β cells of Nf-ya βKO mice could not respond to high glucose stimulation,and the ability of insulin secretion was impaired.In Nf-ya βKO β cells,Soluble N-ethyl-maleimide-sensitive fusion protein(NSF)attachment protein receptor(SNARE),the cell membrane depolarization pathway induced by potassium calcium signaling pathway,calcium ion flow and secretion of insulin under the stimulation of potassium ions were not affected.6.Both m RNA expression and protein level of GLUT2 in Nf-ya βKO islets were significantly decreased,and GLUT2-mediated glucose absorption capacity was decreased.Overexpression of NF-Y in vitro enhanced GLUT2 expression and glucose absorption capacity in NIT1 cells,suggesting that the loss of NF-YA leads to an impairment of GLUT2-mediated glucose absorption.Further evidence indicated that there were three CCAAT boxes of NF-YA binding motif in the Glut2 promoter.The site-directed mutation assay showed that NF-YA directly regulated Glut2 transcription.7.In contrast to the enhanced mitochondrial function in Nf-ya βKO islet under basal glucose concentration,which was striking decreased under high glucose stimulation.The expression of mitochondrial oxidative phosphorylation related genes αS9,GR1,Cyct·C,COX3,COX4,COX5 and ATP synthase gene ATP5α,ATP5β,ATP5C1 were significantly decreased.Moreover,the mitochondrial ultrastructure of β-cells was damaged in Nf-ya βKO mice.Both the expression of mitochondrial proliferation gene Pgc1α and the number of mitochondria were increased.Conclusion: This study showed that NF-Y played an important role in the maturation,development and function of islet β cells and islet structure maintenance in mice.1.The specific loss of NF-Y in mice β cells led to the decrease of islet size and the number of β-cells,decreased insulin synthesis and insulin secretion,and the impairment of glucose homeostasis.2.NF-Y regulates β-cells proliferation,development and maturation by modulating the cytoskeleton.3.NF-YA deletion in mice β cells attenuated glucose absorption and insulin secretion by regulating GLUT2 expression and mitochondrial function. |
PDF Full Text Request