Involvement of conventional kinesin in glucose-stimulated secretory granule movements and exocytosis in clonal pancreatic beta-cells.
Recruitment of secretory vesicles to the cell surface is essential for the sustained secretion of insulin in response to glucose. At present, the molecular motors involved in this movement, and the mechanisms whereby they may be regulated, are undefined. To investigate the role of kinesin family members, we labelled densecore vesicles in clonal beta-cells using an adenovirally expressed, vesicle-targeted green fluorescent protein (phogrin.EGFP), and employed immunoadsorption to obtain highly purified insulin-containing vesicles. Whereas several kinesin family members were expressed in this cell type, only conventional kinesin heavy chain (KHC) was detected in vesicle preparations. Expression of a dominant-negative KHC motor domain (KHC(mut)) blocked all vesicular movements with velocity >0.4 micro m second(-1), which demonstrates that kinesin activity was essential for vesicle motility in live beta-cells. Moreover, expression of KHC(mut) strongly inhibited the sustained, but not acute, stimulation of secretion by glucose. Finally, vesicle movement was stimulated by ATP dose-dependently in permeabilized cells, which suggests that glucose-induced increases in cytosolic [ATP] mediate the effects of the sugar in vivo, by enhancing kinesin activity. These data therefore provide evidence for a novel mechanism whereby glucose may enhance insulin release.
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D002999:Clone Cells / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005089:Exocytosis / Q000187:drug effects / Q000235:genetics; D005947:Glucose / Q000378:metabolism* / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins; D008565:Membrane Proteins; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics*; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D011993:Recombinant Fusion Proteins; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism*
D007328:Insulin; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; C102889:PTPRN2 protein, human; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D016547:Kinesins; D005947:Glucose