The heterotrimeric G-protein Gi is localized to the insulin secretory granules of beta-cells and is involved in insulin exocytosis.
Mastoparan, a tetradecapeptide found in wasp venom that stimulates G-proteins, increases insulin secretion from beta-cells. In this study, we have examined the role of heterotrimeric G-proteins in mastoparan-induced insulin secretion from the insulin-secreting beta-cell line beta-TC3. Mastoparan stimulated insulin secretion in a dose-dependent manner from digitonin-permeabilized beta-TC3 cells. Active mastoparan analogues mastoparan 7, mastoparan 8, and mastoparan X also stimulated secretion. Mastoparan 17, an inactive analogue of mastoparan, did not increase insulin secretion from permeabilized beta-TC3 cells. Mastoparan-induced insulin secretion from permeabilized beta-TC3 cells was inhibited by pretreatment of the cells with pertussis toxin, suggesting that mastoparan-induced insulin secretion is mediated through a pertussis toxin-sensitive G-protein present distally in exocytosis. Enriched insulin secretory granules (ISG) were prepared by sucrose/nycodenz ultracentrifugation. Western immunoblotting performed on beta-TC3 homogenate and ISG demonstrated that G alpha i was dramatically enriched in ISG. Levels of G alpha o and G alpha q were comparable in homogenate and ISG. Mastoparan stimulated ISG GTPase activity in a pertussis toxin-sensitive manner. Mastoparan 7 and mastoparan 8 also stimulated GTPase activity in the ISG, while the inactive analogue mastoparan 17 had no effect. Selective localization of G alpha i to ISG was confirmed with electron microscopic immunocytochemistry in beta-TC3 cells and beta-cells from rat pancreas. In contrast to G alpha o and G alpha q, G alpha was clearly localized to the ISG. Together, these data suggest that mastoparan may act through the heterotrimeric G-protein G alpha i located in the ISG of beta-cells to stimulate insulin secretion.
Konrad
R J
RJ
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Young
R A
RA
Record
R D
RD
Smith
R M
RM
Butkerait
P
P
Manning
D
D
Jarett
L
L
Wolf
B A
BA
D000818:Animals; D015153:Blotting, Western; D002463:Cell Membrane Permeability; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004072:Digitonin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005089:Exocytosis; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D007340:Insulinoma; D036341:Intercellular Signaling Peptides and Proteins; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D010455:Peptides; D037342:Pertussis Toxin; D051381:Rats; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014862:Wasp Venoms / Q000737:chemistry / Q000494:pharmacology
D007328:Insulin; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D010566:Virulence Factors, Bordetella; D014862:Wasp Venoms; C037500:mastoparan; D037342:Pertussis Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D004072:Digitonin