Exendin-4 differentiation of a human pancreatic duct cell line into endocrine cells: involvement of PDX-1 and HNF3beta transcription factors

J Cell Physiol. 2002 Sep;192(3):304-14. doi: 10.1002/jcp.10143.

Abstract

Exendin-4 (EX-4), a long acting agonist of GLP-1, induces an endocrine phenotype in Capan-1 cells. Under culture conditions which include serum, approximately 10% of the cells contain insulin and glucagon. When exposed to EX-4 (0.1 nM, up to 5 days), the number of cells containing insulin and glucagon increased to approximately 40%. Western blot analysis detected a progressive increase in protein levels of glucokinase and GLUT2 over 3 days of EX-4 treatment. We explored the sequence of activation of certain transcription factors known to be essential for the beta cell phenotype: PDX-1, Beta2/NeuroD, and hepatocyte nuclear factor 3beta (HNF3beta). Double immunostaining showed that PDX-1 coexisted with insulin and glucagon in EX-4-treated cells. Treatment caused an increase in PDX-1 protein levels by 24 h and induced its nuclear translocation. Beta2/NeuroD protein levels also increased progressively over 24 h. HNF3beta protein level increased twofold as early as 6 h after EX-4 treatment. EMSA results indicated that EX-4 caused a 12-fold increase in HNF3beta binding to PDX-1 promoter area II. Beta2/NeuroD protein levels progressively increased after 24 h treatment. Differentiation to insulin-producing cells was also seen when Capan-1 cells were transfected with pdx-1, with 80% of these cells expressing insulin 3 days after transfection. PDX-1 antisense totally inhibited such conversion. During the differentiation of duct cells to endocrine cells, cAMP levels (EX-4 is a ligand for the GLP-1, G-protein coupled receptor) and MAP kinase activity increased. Our results indicate that EX-4 activates adenylyl cyclase and MAP kinase which, in turn, may lead to activation of transcription factors necessary for an endocrine phenotype.

MeSH terms

  • Cell Differentiation / drug effects
  • Cell Line
  • Cyclic AMP / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Exenatide
  • Glucagon / agonists
  • Glucagon / metabolism
  • Glucagon-Like Peptide 1
  • Glucagon-Like Peptide-1 Receptor
  • Hepatocyte Nuclear Factor 3-beta
  • Homeodomain Proteins*
  • Humans
  • Insulin / metabolism
  • Islets of Langerhans / cytology*
  • Islets of Langerhans / drug effects*
  • Islets of Langerhans / metabolism
  • Mitogen-Activated Protein Kinases / metabolism
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Pancreatic Ducts / cytology*
  • Pancreatic Ducts / drug effects*
  • Pancreatic Ducts / metabolism
  • Peptide Fragments / agonists
  • Peptides / pharmacology*
  • Promoter Regions, Genetic
  • Protein Precursors / agonists
  • Receptors, Glucagon / metabolism
  • Trans-Activators / genetics
  • Trans-Activators / metabolism*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transfection
  • Venoms*

Substances

  • DNA-Binding Proteins
  • FOXA2 protein, human
  • GLP1R protein, human
  • Glucagon-Like Peptide-1 Receptor
  • Homeodomain Proteins
  • Insulin
  • Nuclear Proteins
  • Peptide Fragments
  • Peptides
  • Protein Precursors
  • Receptors, Glucagon
  • Trans-Activators
  • Transcription Factors
  • Venoms
  • pancreatic and duodenal homeobox 1 protein
  • Hepatocyte Nuclear Factor 3-beta
  • Glucagon-Like Peptide 1
  • Glucagon
  • Exenatide
  • Cyclic AMP
  • Mitogen-Activated Protein Kinases