Arf nucleotide binding site opener [ARNO] promotes sequential activation of Arf6, Cdc42 and Rac1 and insulin secretion in INS 832/13 β-cells and rat islets.
Glucose-stimulated insulin secretion [GSIS] involves interplay between small G-proteins and their regulatory factors. Herein, we tested the hypothesis that Arf nucleotide binding site opener [ARNO], a guanine nucleotide-exchange factor [GEF] for the small G-protein Arf6, mediates the functional activation of Arf6, and that ARNO/Arf6 signaling axis, in turn, controls the ... activation of Cdc42 and Rac1, which have been implicated in GSIS. Molecular biological [i.e., expression of inactive mutants or siRNA] and pharmacological approaches were employed to assess the roles for ARNO/Arf6 signaling pathway in insulin secretion in normal rat islets and INS 832/13 cells. Degrees of activation of Arf6 and Cdc42/Rac1 were quantitated by GST-GGA3 and PAK-1 kinase pull-down assays, respectively. ARNO is expressed in INS 832/13 cells, rat islets and human islets. Expression of inactive mutants of Arf6 [Arf6-T27N] or ARNO [ARNO-E156K] or siRNA-ARNO markedly reduced GSIS in isolated β-cells. SecinH3, a selective inhibitor of ARNO/Arf6 signaling axis, also inhibited GSIS in INS 832/13 cells and rat islets. Stimulatory concentrations of glucose promoted Arf6 activation, which was inhibited by secinH3 or siRNA-ARNO, suggesting that ARNO/Arf6 signaling cascade is necessary for GSIS. SecinH3 or siRNA-ARNO also inhibited glucose-induced activation of Cdc42 and Rac1 suggesting that ARNO/Arf6 might be upstream to Cdc42 and Rac1 activation steps, which are necessary for GSIS. Lastly, co-immunoprecipitation and confocal microscopic studies suggested increased association between Arf6 and ARNO in glucose-stimulated β-cells. These findings provide the first evidence to implicate ARNO in the sequential activation of Arf6, Cdc42 and Rac1 culminating in GSIS.
Mesh Terms:
ADP-Ribosylation Factors, Animals, Cell Culture Techniques, Cell Line, Fluorescent Antibody Technique, GTPase-Activating Proteins, Glucose, Humans, Hydrophobic and Hydrophilic Interactions, Immunoprecipitation, Insulin, Insulin-Secreting Cells, Islets of Langerhans, Male, Microscopy, Confocal, RNA, Small Interfering, Rats, Rats, Sprague-Dawley, Transfection, cdc42 GTP-Binding Protein, rac1 GTP-Binding Protein
ADP-Ribosylation Factors, Animals, Cell Culture Techniques, Cell Line, Fluorescent Antibody Technique, GTPase-Activating Proteins, Glucose, Humans, Hydrophobic and Hydrophilic Interactions, Immunoprecipitation, Insulin, Insulin-Secreting Cells, Islets of Langerhans, Male, Microscopy, Confocal, RNA, Small Interfering, Rats, Rats, Sprague-Dawley, Transfection, cdc42 GTP-Binding Protein, rac1 GTP-Binding Protein
Biochem. Pharmacol.
Date: Apr. 15, 2011
PubMed ID: 21276423
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