The Rap-B-Raf signalling pathway is activated by glucose and glucagon-like peptide-1 in human islet cells.
OBJECTIVE
Glucose and glucagon-like peptide-1 have been shown to activate extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase in beta cells. We examined the contributions of the small GTPases Rap and Ras and the serine-threonine kinases B-Raf and Raf-1 to the activation of these kinases in human islet cells.


METHODS
The expression of Rap, Ras, B-Raf and Raf-1 in human islets was examined by immunohistochemistry and immunoblotting. Human islets were incubated in glucose at concentrations of 2.5 and 15 mmol/l and were stimulated with 10 nmol/l glucagon-like peptide-1. The activation of ERK and Raf kinases was examined by phosphorylation-specific antibodies and immuno-complexed kinase assays. The activation of Rap and Ras was determined by pull-down assays. Stimulation of phosphoinositide 3-kinase was detected by immuno-complexed lipid kinase assays.


RESULTS
Extracellular-regulated kinase and protein kinase B (a downstream target of phosphoinositide 3-kinase) were activated in islets stimulated with glucose and glucagon-like peptide-1. In these islets, the Rap-B-Raf signalling pathway was activated preferentially compared with Ras and Raf-1, and activated Rap and B-Raf mediated ERK stimulation in kinase assays in vitro. In addition, Rap rather than Ras mediated activation of phosphoinositide 3-kinase in islets stimulated with glucose and glucagon-like peptide-1.


CONCLUSIONS
In human islet cells, glucose and glucagon-like peptide-1 activate the Rap and B-Raf signalling module, which mediates ERK activation in assays in vitro. Rap also activates phosphoinositide 3-kinase, delineating central roles for Rap and B-Raf as therapeutic targets for beta cell growth in diabetes mellitus.
Trümper
J
J
Department of Internal Medicine, Division of Gastroenterology and Endocrinology, Philipps University of Marburg, Germany.
Ross
D
D
Jahr
H
H
Brendel
M D
MD
Göke
R
R
Hörsch
D
D
D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D016336:Gene Products, vpr / Q000502:physiology*; D005934:Glucagon / Q000494:pharmacology*; D052216:Glucagon-Like Peptide 1; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011498:Protein Precursors / Q000494:pharmacology*; D048493:Proto-Oncogene Proteins B-raf / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects*; D048490:raf Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D004791:Enzyme Inhibitors; D048049:Extracellular Signal-Regulated MAP Kinases; D020558:GTP Phosphohydrolases; D016336:Gene Products, vpr; D005934:Glucagon; D052216:Glucagon-Like Peptide 1; D005947:Glucose; D010446:Peptide Fragments; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011498:Protein Precursors; D048493:Proto-Oncogene Proteins B-raf; D048490:raf Kinases; C482119:BRAF protein, human