title: 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.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University and β-Cell Biochemistry Laboratory, John D. Dingell VA Medical Center, 259 Mack Avenue, Detroit, MI 48201, USA.
authors.forename: Bhavaani
authors.initials: B
authors.lastname: Jayaram
authors.forename: Ismail
authors.initials: I
authors.lastname: Syed
authors.forename: Chandrashekara N
authors.initials: CN
authors.lastname: Kyathanahalli
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Rhodes
authors.forename: Anjaneyulu
authors.initials: A
authors.lastname: Kowluru
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000502:physiology*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D047468:Immunoprecipitation; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism*; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
chemical_list: D000089982:ADP-Ribosylation Factor 6; D020690:GTPase-Activating Proteins; D007328:Insulin; D034741:RNA, Small Interfering; C102474:cytohesin-2; C499134:Rac1 protein, rat; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; C000715349:Arf6 protein, rat; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D005947:Glucose