Suppression of Pdx-1 perturbs proinsulin processing, insulin secretion and GLP-1 signalling in INS-1 cells

Diabetologia. 2005 Apr;48(4):720-31. doi: 10.1007/s00125-005-1692-8. Epub 2005 Mar 9.

Abstract

Aims/hypothesis: Mutations in genes encoding HNF-4alpha, HNF-1alpha and IPF-1/Pdx-1 are associated with, respectively, MODY subtypes-1, -3 and -4. Impaired glucose-stimulated insulin secretion is the common primary defect of these monogenic forms of diabetes. A regulatory circuit between these three transcription factors has also been suggested. We aimed to explore how Pdx-1 regulates beta cell function and gene expression patterns.

Methods: We studied two previously established INS-1 stable cell lines permitting inducible expression of, respectively, Pdx-1 and its dominant-negative mutant. We used HPLC for insulin processing, adenovirally encoded aequorin for cytosolic [Ca2+], and transient transfection of human growth hormone or patch-clamp capacitance recordings to monitor exocytosis.

Results: Induction of DN-Pdx-1 resulted in defective glucose-stimulated and K+-depolarisation-induced insulin secretion in INS-1 cells, while overexpression of Pdx-1 had no effect. We found that DN-Pdx-1 caused down-regulation of fibroblast growth factor receptor 1 (FGFR1), and consequently prohormone convertases (PC-1/3 and -2). As a result, DN-Pdx-1 severely impaired proinsulin processing. In addition, induction of Pdx-1 suppressed the expression of glucagon-like peptide 1 receptor (GLP-1R), which resulted in marked reduction of both basal and GLP-1 agonist exendin-4-stimulated cellular cAMP levels. Induction of DN-Pdx-1 did not affect glucokinase activity, glycolysis, mitochondrial metabolism or ATP generation. The K+-induced cytosolic [Ca2+] rise and Ca2+-evoked exocytosis (membrane capacitance) were not abrogated.

Conclusions/interpretation: The severely impaired proinsulin processing combined with decreased GLP-1R expression and cellular cAMP content, rather than metabolic defects or altered exocytosis, may contribute to the beta cell dysfunction induced by Pdx-1 deficiency.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Animals
  • Calcium Signaling / physiology
  • Cell Line, Tumor
  • Cyclic AMP / metabolism
  • Dose-Response Relationship, Drug
  • Doxycycline / pharmacology
  • Exocytosis / physiology
  • Gene Expression / drug effects
  • Gene Expression / genetics
  • Gene Expression Regulation, Neoplastic / drug effects
  • Glucagon-Like Peptide-1 Receptor
  • Glucokinase / genetics
  • Glucose / metabolism
  • Glucose / pharmacology
  • Glycolysis
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • Homeodomain Proteins / physiology*
  • Human Growth Hormone / genetics
  • Human Growth Hormone / metabolism
  • Insulin / metabolism*
  • Insulin Secretion
  • Islets of Langerhans / drug effects
  • Islets of Langerhans / metabolism
  • Mitochondria / metabolism
  • Mutation
  • Proinsulin / metabolism*
  • Proprotein Convertases / genetics
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Rats
  • Receptor Protein-Tyrosine Kinases / genetics
  • Receptor, Fibroblast Growth Factor, Type 1
  • Receptors, Fibroblast Growth Factor / genetics
  • Receptors, Glucagon / genetics
  • Receptors, Glucagon / physiology*
  • Signal Transduction / physiology*
  • Time Factors
  • Trans-Activators / genetics
  • Trans-Activators / metabolism
  • Trans-Activators / physiology*
  • Transfection

Substances

  • GLP1R protein, human
  • Glp1r protein, rat
  • Glucagon-Like Peptide-1 Receptor
  • Homeodomain Proteins
  • Insulin
  • RNA, Messenger
  • Receptors, Fibroblast Growth Factor
  • Receptors, Glucagon
  • Trans-Activators
  • pancreatic and duodenal homeobox 1 protein
  • Human Growth Hormone
  • Adenosine Triphosphate
  • Proinsulin
  • Cyclic AMP
  • Glucokinase
  • Fgfr1 protein, rat
  • Receptor Protein-Tyrosine Kinases
  • Receptor, Fibroblast Growth Factor, Type 1
  • Proprotein Convertases
  • Glucose
  • Doxycycline