Mastoparan-Induced Insulin Secretion from Insulin-Secreting βTC3 and INS-1 Cells: Evidence for Its Regulation by Rho Subfamily of G Proteins

• Published in: Endocrinology (Philadelphia) Vol. 144; no. 10; pp. 4508 - 4518
• Main Authors: Amin, Rajesh H, Chen, Hai-Qing, Veluthakal, Rajakrishnan, Silver, Robert B, Li, Jingsong, Li, GuoDong, Kowluru, Anjaneyulu
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Endocrine Society 01.10.2003
• Subjects: Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Vertebrates: endocrinology; Pancreatic hormone; Insulin; G protein; Endocrine secretion
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/en.2003-0106

Mastoparan, a tetradecapeptide from wasp venom, stimulates insulin secretion from the islet β-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 βTC3 and INS-1 cells. Preincubation of βTC3 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 β-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.