title: Mastoparan-induced insulin secretion from insulin-secreting betaTC3 and INS-1 cells: evidence for its regulation by Rho subfamily of G proteins.
abstract: Mastoparan, a tetradecapeptide from wasp venom, stimulates insulin secretion from the islet beta-cells, presumably via activation of trimeric G proteins. Herein, we used Clostridial toxins, which selectively modify and inactivate the Rho subfamily of G proteins, to examine whether mastoparan-induced insulin secretion also involves activation of these signaling proteins. Mastoparan, but not mastoparan 17 (an inactive analog of mastoparan), significantly stimulated insulin secretion from betaTC3 and INS-1 cells. Preincubation of betaTC3 cells with either Clostridium difficille toxin B, which inactivates Rho, Cdc42, and Rac, or Clostridium sordellii toxin, which inactivates Ras, Rap, and Rac, markedly attenuated the mastoparan-induced insulin secretion, implicating Rac in this phenomenon. Mastoparan-stimulated insulin secretion was resistant to GGTI-2147, a specific inhibitor of geranylgeranylation of Rho G proteins (e.g. Rac), suggesting that mastoparan induces direct activation of Rac via GTP/GDP exchange. This was confirmed by a pull-down assay that quantifies the binding of activated (i.e. GTP-bound) Rac to p21-activated kinase. However, glucose-induced insulin secretion from these cells was abolished by toxin B or GGTI-2147, suggesting that the geranylgeranylation step is critical for glucose-stimulated secretion. Mastoparan significantly increased the translocation of cytosolic Rac and Cdc42 to the membrane fraction. Confocal light microscopy revealed a substantial degree of colocalization of Rac (and, to a lesser degree, Cdc42) with insulin in beta-cells exposed to mastoparan. Further, stable expression of a dominant negative (N17Rac) form of Rac into INS-1 cells resulted in a significant reduction in mastoparan-stimulated insulin secretion from these cells. Taken together, our findings implicate Rho G proteins, specifically Rac, in mastoparan-induced insulin release.
authors.affiliation: Department of Pharmaceutical Sciences, Wayne State University, Detroit, Michigan 48201, USA.
authors.forename: Rajesh H
authors.initials: RH
authors.lastname: Amin
authors.forename: Hai-Qing
authors.initials: HQ
authors.lastname: Chen
authors.forename: Rajakrishnan
authors.initials: R
authors.lastname: Veluthakal
authors.forename: Robert B
authors.initials: RB
authors.lastname: Silver
authors.forename: Jingsong
authors.initials: J
authors.lastname: Li
authors.forename: GuoDong
authors.initials: G
authors.lastname: Li
authors.forename: Anjaneyulu
authors.initials: A
authors.lastname: Kowluru
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D005799:Genes, Dominant; D005947:Glucose / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D036341:Intercellular Signaling Peptides and Proteins; D007515:Islets of Langerhans / Q000378:metabolism*; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008566:Membranes / Q000378:metabolism; D051379:Mice; D010455:Peptides; D017368:Protein Prenylation / Q000187:drug effects; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution; D014862:Wasp Venoms / Q000494:pharmacology*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000502:physiology; D020741:rho GTP-Binding Proteins / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; C466792:GGTI 2147; D007093:Imidazoles; D007328:Insulin; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014862:Wasp Venoms; C072292:lethal toxin LT, Clostridium sordellii; C031815:toxB protein, Clostridium difficile; C037500:mastoparan; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D007930:Leucine; D005947:Glucose; D002118:Calcium