Novel Regulators of Islet Beta-Cell Function in Health and Diabetes

 描述(由申请人提供)： 我们实验室的原创贡献为小G蛋白确立了新的作用[例如， 在正常啮齿动物和人类胰岛的葡萄糖刺激的胰岛素分泌[GSIS]中。我们提供了第一个证据，表明这些G-蛋白通过在其C-末端加入法尼基或香叶基而发生翻译后脂肪作用。 我们实验室最近的研究表明，蛋白法尼基转移酶[FTase]和香叶基香叶基转移酶[GGTase]在包括GSIS在内的ç细胞功能中具有新的调节作用。我们的努力也导致了对这些G蛋白的新的调节因子的识别，包括GDP解离抑制物[GDIs]和鸟嘌呤核苷酸交换因子[GEF]，它们似乎精确地控制了导致GSIS的信号步骤。在这些研究过程中，我们注意到在糖毒性、内质网应激和T2 DM的体外和体内模型中，FTase/GGTase信号通路存在明显的缺陷。作为先前资助的研究的合乎逻辑的扩展，并基于最近积累的令人兴奋的初步证据，我们建议检验总体假设，即糖毒和内质网应激条件诱导FTase/GGTase信号级联中的缺陷，导致小G蛋白[rac1]的持续激活和线粒体失调和胰岛？细胞的死亡。我们将通过在三个具体目标下描述的实验来实现这一目标。在目标1中，我们将确定糖毒和内质网应激条件是否促进FTase/GGTase信号通路的改变，从而导致胰岛？细胞的代谢功能障碍和凋亡。作为支持，我们提出了初步证据，表明在糖毒条件下，胰腺细胞中FTase/GGTase活性显著降低，小G蛋白[rac1]持续激活。Aim 2中的研究将阐明在糖毒和内质网应激条件下，胰岛细胞中G蛋白的持续激活和加速应激激酶信号转导步骤[JNK1/2，p38 MAP Kinase]的机制。我们的初步证据还表明，Tiam1[NSC23766]和Vav2[EHop-016]的抑制剂，这两个已知的rac1的GEF，显著减弱了胰岛？细胞的糖毒效应。AIM 3中描述的实验将确定药物干预和预防FTase/GGTase信号轴的改变、G蛋白的持续[结构性]激活以及下游代谢事件的异常是否能恢复饮食诱导肥胖和T2 DM动物模型的正常细胞功能。建议的研究是创新的，具有显著的翻译影响，因为它们将为导致T2 DM胰岛功能障碍的FTase/GGTase信号缺陷事件的潜在机制提供新的见解，并且这些研究获得的数据将成为识别新靶点的基础 在FTase/GGTase和G蛋白中，信号级联作用于治疗人类T2 DM的治疗方法的发展。我们在包括GSIS在内的胰岛功能的G蛋白预酯化领域的长期专业知识为我们提供了一个独特的机会来解决胰岛功能在健康和糖尿病中的这些重要方面。

 DESCRIPTION (provided by applicant): Original contributions from our laboratory have established novel roles for small G-proteins [e.g., Rac1, Cdc42] in glucose-stimulated insulin secretion [GSIS] from normal rodent and human islets. We provided the first evidence to suggest that these G-proteins undergo post-translational lipidation via incorporation of farnesyl or geranylgeranyl groups at their C-termini. Recent studies from our laboratory have demonstrated novel regulatory roles for protein farnesyltransferase [FTase] and geranylgeranyltransferase [GGTase] in ß-cell function including GSIS. Our efforts have also led to the identification of novel regulatory factors for these G-proteins including GDP-dissociation inhibitors [GDIs] and guanine nucleotide exchange factors [GEFs], which appear to precisely control signaling steps leading to GSIS. During the course of these investigations, we noticed significant defects in FTase/GGTase signaling pathways in in vitro and in vivo models of glucolipotoxicity, endoplasmic reticulum [ER] stress and T2DM. As a logical extension of the previously funded studies, and based on recently accrued exciting preliminary evidence we propose to test the overall hypothesis that glucolipotoxic and ER stress conditions induce defects in the FTase/GGTase signaling cascade leading to sustained activation of small G-proteins [Rac1] and mitochondrial dysregulation and demise of the islet ß-cell. We will accomplish this goal via experiments described under three Specific Aims. In Aim 1, we will determine if glucolipotoxic and ER stress conditions promote alterations in the FTase/GGTase signaling pathway leading to the metabolic dysfunction and apoptosis of the islet ß-cell. In its support, we present preliminary evidence to suggest significant reduction of FTase/GGTase activity and sustained activation of small G-proteins [Rac1] in pancreatic ß-cells under glucolipotoxic conditions. Studies in Aim 2 will delineate the mechanisms underlying sustained activation of G-proteins and accelerated stress kinase signaling steps [JNK1/2, p38 MAP kinase] in the islet ß-cell under conditions of glucolipotoxicity and ER stress. Our preliminary evidence also suggests that inhibitors of Tiam1 [NSC23766] and Vav2 [Ehop-016], two known GEFs for Rac1, significantly attenuated glucotoxic effects in islet ß-cells. Experiments described under Aim 3 will determine if pharmacological intervention and prevention of alterations in FTase/GGTase signaling axis, sustained [constitutive] activation of G-proteins, and abnormalities in downstream metabolic events restore normal ß-cell function in animal models of diet-induced obesity and T2DM. The proposed studies are innovative and carry significant translational impact as they will provide novel insights into the underlying mechanisms for defective FTase/GGTase signaling events leading to islet dysfunction in T2DM, and the data accrued from these studies will form the basis for the identification of novel targets in the FTase/GGTase and G-protein signaling cascades for development of therapeutics to treat T2DM in humans. Our long-standing expertise in the area of G-protein prenylation in islet function including GSIS gives us a unique opportunity to address these important aspects of islet function in health and in diabetes.