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Université de Bordeaux, Cell Biology Program [JE2390 (M.D., B.R., D.H., J.L.) and Institut National de la Santé et de la Recherche Médicale (INSERM) E347 (P.V.)], European Institute of Chemistry and Biology, 33607 Pessac, France; INSERM ERIT-M 0106 (B.V., J.K.-C., F.P.), Cellular Therapy of Diabetes, 59045 Lille, France; and The Nutrition Department (N.M.-M.), The University of Tennessee, Knoxville, Tennessee 37996-1920
Address all correspondence and requests for reprints to: Jochen Lang, Institut Européen de Chimie et Biologie, 2 rue Robert Escarpit, F-33607 Pessac, France. E-mail: j.lang{at}iecb.u-bordeaux.fr.
Prolonged exposure of ß-cells to high glucose (glucotoxicity) diminishes insulin secretion in response to glucose and has been linked to altered generation of metabolism-secretion coupling factors. We have investigated whether glucotoxicity may also alter calcium handling and late steps in secretion such as exocytosis. Clonal INS-1E ß-cells cultured at high glucose (20 or 30 mM vs. 5.5 mM) for 72 h exhibited elevated basal intracellular calcium ([Ca2+]i), which was KATP-channel dependent and due to long-term activation of protein kinase A. An increased amplitude and shortened duration of depolarization-evoked rises in [Ca2+]i were apparent. These changes were probably linked to the observed increased filling of intracellular stores and to short-term activation of protein kinase A. Insulin secretion was reduced not only by acute stimulation with either glucose or KCl but more importantly by direct calcium stimulation of permeabilized cells. These findings indicate a defect in the final steps of exocytosis. To confirm this, we measured expression levels of some 30 proteins implicated in trafficking/exocytosis of post-Golgi vesicles. Several proteins required for calcium-induced exocytosis of secretory granules were down-regulated, such as the soluble N-ethylmaleimide-sensitive factor-sensitive factor attachment receptor (SNARE) proteins VAMP-2 [vesicle (v)-SNARE, vesicle-associated membrane protein 2] and syntaxin 1 as well as complexin. VAMP-2 was also reduced in human islets. In contrast, cell immunostaining and expression levels of several fluorescent proteins suggested that other post-trans-Golgi trafficking steps and compartments are preserved and that cells were not degranulated. Thus, these studies indicate that, in addition to known metabolic changes, glucotoxicity impedes generation of signals for secretion and diminishes the efficiency of late steps in exocytosis.
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