Suppression of Pdx-1 perturbs proinsulin processing, insulin secretion and GLP-1 signalling in INS-1 cells.
OBJECTIVE
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
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.
Wang
H
H
Department of Cell Physiology and Metabolism, University Medical Center, 1211 Geneva 4, Switzerland. Haiyan.Wang@medicine.unige.ch
Iezzi
M
M
Theander
S
S
Antinozzi
P A
PA
Gauthier
B R
BR
Halban
P A
PA
Wollheim
C B
CB
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D020013:Calcium Signaling / Q000502:physiology; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology; D005089:Exocytosis / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D000067757:Glucagon-Like Peptide-1 Receptor; D005941:Glucokinase / Q000235:genetics; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D006019:Glycolysis; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D019382:Human Growth Hormone / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation; D011384:Proinsulin / Q000378:metabolism*; D043484:Proprotein Convertases / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics; D018027:Receptors, Glucagon / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
C000593833:GLP1R protein, human; C000593835:Glp1r protein, rat; D000067757:Glucagon-Like Peptide-1 Receptor; D018398:Homeodomain Proteins; D007328:Insulin; D012333:RNA, Messenger; D017468:Receptors, Fibroblast Growth Factor; D018027:Receptors, Glucagon; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D019382:Human Growth Hormone; D000255:Adenosine Triphosphate; D011384:Proinsulin; D000242:Cyclic AMP; D005941:Glucokinase; C496350:Fgfr1 protein, rat; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1; D043484:Proprotein Convertases; D005947:Glucose; D004318:Doxycycline