Konrad R J, Young R A, Record R D, Smith R M, Butkerait P, Manning D, Jarett L, Wolf B A
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
J Biol Chem. 1995 May 26;270(21):12869-76. doi: 10.1074/jbc.270.21.12869.
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.
蜂毒肽是一种在黄蜂毒液中发现的十四肽，可刺激G蛋白，增加β细胞的胰岛素分泌。在本研究中，我们研究了异源三聚体G蛋白在蜂毒肽诱导胰岛素分泌细胞系β-TC3分泌胰岛素中的作用。蜂毒肽以剂量依赖的方式刺激经洋地黄皂苷通透处理的β-TC3细胞分泌胰岛素。活性蜂毒肽类似物蜂毒肽7、蜂毒肽8和蜂毒肽X也能刺激分泌。蜂毒肽17是蜂毒肽的无活性类似物，不能增加通透处理的β-TC3细胞的胰岛素分泌。用百日咳毒素预处理细胞可抑制蜂毒肽诱导的通透处理的β-TC3细胞分泌胰岛素，这表明蜂毒肽诱导的胰岛素分泌是通过位于胞吐作用远端的对百日咳毒素敏感的G蛋白介导的。通过蔗糖/尼可登超速离心制备了富集的胰岛素分泌颗粒（ISG）。对β-TC3匀浆和ISG进行的蛋白质免疫印迹分析表明，Gαi在ISG中显著富集。Gαo和Gαq在匀浆和ISG中的水平相当。蜂毒肽以百日咳毒素敏感的方式刺激ISG的GTP酶活性。蜂毒肽7和蜂毒肽8也能刺激ISG中的GTP酶活性，而无活性类似物蜂毒肽1则没有作用。通过电子显微镜免疫细胞化学在β-TC3细胞和大鼠胰腺β细胞中证实了Gαi在ISG中的选择性定位。与Gαo和Gαq不同，Gαi明显定位于ISG。这些数据共同表明，蜂毒肽可能通过位于β细胞ISG中的异源三聚体G蛋白Gαi发挥作用，以刺激胰岛素分泌。
