Silva Kelly E, Barbosa Helena C, Rafacho Alex, Bosqueiro José R, Stoppiglia Luiz F, Carneiro Everardo M, Borelli Maria I, Del Zotto Hector, Gagliardino Juan J, Boschero Antonio C
Departamento de Fisiologia e Biofísica, Instituto de Biologia Universidade Estadual de Campinas, 13083-970, Campinas-SP, Brazil.
Regul Pept. 2008 Jun 5;148(1-3):39-45. doi: 10.1016/j.regpep.2008.02.008. Epub 2008 Mar 4.
Islet Neogenesis Associated Protein (INGAP) increases pancreatic beta-cell mass and potentiates glucose-induced insulin secretion. Here, we investigated the effects of the pentadecapeptide INGAP-PP in adult cultured rat islets upon the expression of proteins constitutive of the K(+)(ATP) channel, Ca(2+) handling, and insulin secretion. The islets were cultured in RPMI medium with or without INGAP-PP for four days. Thereafter, gene (RT-PCR) and protein expression (Western blotting) of Foxa2, SUR1 and Kir6.2, cytoplasmic Ca(2+) (Ca(2+)), static and dynamic insulin secretion, and (86)Rb efflux were measured. INGAP-PP increased the expression levels of Kir6.2, SUR1 and Foxa2 genes, and SUR1 and Foxa2 proteins. INGAP-PP cultured islets released significantly more insulin in response to 40 mM KCl and 100 muM tolbutamide. INGAP-PP shifted to the left the dose-response curve of insulin secretion to increasing concentrations of glucose (EC(50) of 10.0+/-0.4 vs. 13.7+/-1.5 mM glucose of the controls). It also increased the first phase of insulin secretion elicited by either 22.2 mM glucose or 100 microM tolbutamide and accelerated the velocity of glucose-induced reduction of (86)Rb efflux in perifused islets. These effects were accompanied by a significant increase in Ca(2+) and the maintenance of a considerable degree of Ca(2+) oscillations. These results confirm that the enhancing effect of INGAP-PP upon insulin release, elicited by different secretagogues, is due to an improvement of the secretory function in cultured islets. Such improvement is due, at least partly, to an increased K(+)(ATP) channel protein expression and/or changing in the kinetic properties of these channels and augmented Ca(2+) response. Accordingly, INGAP-PP could potentially be used to maintain the functional integrity of cultured islets and eventually, for the prevention and treatment of diabetes.
胰岛新生相关蛋白（INGAP）可增加胰腺β细胞量，并增强葡萄糖诱导的胰岛素分泌。在此，我们研究了十五肽INGAP - PP对成年大鼠培养胰岛中构成钾离子ATP通道、钙离子处理及胰岛素分泌相关蛋白表达的影响。胰岛在含或不含INGAP - PP的RPMI培养基中培养四天。此后，检测了叉头框蛋白A2（Foxa2）、磺脲类受体1（SUR1）和内向整流钾离子通道亚基6.2（Kir6.2）的基因（逆转录聚合酶链反应）和蛋白表达（蛋白质印迹法）、细胞质钙离子浓度（[Ca²⁺]i）、静态和动态胰岛素分泌以及⁸⁶Rb外流。INGAP - PP增加了Kir6.2、SUR1和Foxa2基因以及SUR1和Foxa2蛋白的表达水平。INGAP - PP培养的胰岛对40 mM氯化钾和100 μM甲苯磺丁脲的反应释放出显著更多的胰岛素。INGAP - PP使胰岛素分泌的剂量反应曲线向左移动，以应对葡萄糖浓度的增加（对照组的半数有效浓度（EC₅₀）为13.7±1.5 mM葡萄糖，INGAP - PP组为10.0±0.4 mM葡萄糖）。它还增加了由22.2 mM葡萄糖或100 μM甲苯磺丁脲引发的胰岛素分泌的第一相，并加快了灌注胰岛中葡萄糖诱导的⁸⁶Rb外流减少的速度。这些效应伴随着[Ca²⁺]i的显著增加以及相当程度的[Ca²⁺]i振荡的维持。这些结果证实，INGAP - PP对不同促分泌剂引发的胰岛素释放的增强作用，是由于培养胰岛分泌功能的改善。这种改善至少部分归因于钾离子ATP通道蛋白表达的增加和/或这些通道动力学特性的改变以及增强的[Ca²⁺]i反应。因此，INGAP - PP有可能用于维持培养胰岛的功能完整性，并最终用于糖尿病的预防和治疗。
