Sustained exposure to high glucose concentrations modifies glucose signaling and the mechanics of secretory vesicle fusion in primary rat pancreatic beta-cells.
The mechanism(s) by which chronic hyperglycemia impairs glucose-stimulated insulin secretion is poorly defined. Here, we compare the "nanomechanics" of single exocytotic events in primary rat pancreatic beta-cells cultured for 48 h at optimal (10 mmol/l) or elevated (30 mmol/l) glucose concentrations. Cargo release was imaged by total internal reflection fluorescence microscopy of lumen-targeted probes (neuropeptide Y [NPY]-pH-insensitive yellow fluorescent protein [NPY-Venus] or NPY-monomeric red fluorescent protein), while the fate of the vesicle membrane was reported simultaneously with phosphatase-on-the-granule-of-insulinoma-enhanced green fluorescent protein. Under all conditions studied, exocytosis proceeded via a "cavity recapture" mechanism in which the vesicle and plasma membranes fused transiently. While essentially complete release of NPY-Venus was observed in 24 +/- 1% of glucose-stimulated exocytotic events in cells maintained at 10 mmol/l glucose, this value was reduced reversibly to 5 +/- 2% of events by culture at 30 mmol/l glucose, in line with decreases in Glut2 and glucokinase gene expression, and attenuated glucose-stimulated increases in NADPH and intracellular [Ca2+]. Since vesicle release in response to cell depolarization with KCl was not affected by culture at 30 mmol/l glucose, we conclude that hyperglycemia causes the abnormal termination of individual insulin release events principally by inhibiting glucose signaling.
Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK.
Takashi
T
Tsuboi
Magalie A
MA
Ravier
Laura E
LE
Parton
Guy A
GA
Rutter
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D017931:DNA Primers; D005089:Exocytosis; D017930:Genes, Reporter; D005947:Glucose / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D018613:Microscopy, Confocal; D009478:Neuropeptide Y / Q000494:pharmacology; D011384:Proinsulin / Q000032:analysis; D011498:Protein Precursors / Q000032:analysis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050857:Synaptotagmins / Q000235:genetics
D017931:DNA Primers; D007328:Insulin; D009478:Neuropeptide Y; D011498:Protein Precursors; D050857:Synaptotagmins; C013737:preproinsulin; D011384:Proinsulin; D005947:Glucose; D002118:Calcium