title: Glucotoxicity inhibits late steps of insulin exocytosis.
abstract: Prolonged exposure of beta-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 beta-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.
authors.affiliation: Université de Bordeaux, Cell Biology Program, and Institut National de la Santé et de la Recherche Médicale E347, Pessac, France.
authors.forename: Mathilde
authors.initials: M
authors.lastname: Dubois
authors.forename: Pierre
authors.initials: P
authors.lastname: Vacher
authors.forename: Benoît
authors.initials: B
authors.lastname: Roger
authors.forename: Deborah
authors.initials: D
authors.lastname: Huyghe
authors.forename: Brigitte
authors.initials: B
authors.lastname: Vandewalle
authors.forename: Julie
authors.initials: J
authors.lastname: Kerr-Conte
authors.forename: François
authors.initials: F
authors.lastname: Pattou
authors.forename: Naima
authors.initials: N
authors.lastname: Moustaïd-Moussa
authors.forename: Jochen
authors.initials: J
authors.lastname: Lang
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002451:Cell Compartmentation; D002478:Cells, Cultured; D005089:Exocytosis / Q000187:drug effects*; D005947:Glucose / Q000633:toxicity*; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D014018:Tissue Distribution; D033921:Vesicular Transport Proteins / Q000378:metabolism
chemical_list: D007328:Insulin; D033921:Vesicular Transport Proteins; D005947:Glucose; D002118:Calcium