Protein Prenyltransferases in Glucose-Stimulated Insulin Secretion

描述（由申请人提供）：新出现的证据表明小G蛋白[例如，Rac 1和Cdc 42]在胰岛细胞中葡萄糖刺激的胰岛素分泌[GSIS]中。然而，葡萄糖介导的这些蛋白质激活的精确细胞机制仍然只是部分了解。使用通用[例如，他汀类药物]，以及更具体的前药[例如，烯丙基或乙烯基法呢醇和香叶基香叶醇]和肽模拟物[例如，GGTI-2147]蛋白质异戊二烯基转移酶[PPT酶]抑制剂，我们最近报道了胰岛G蛋白的C-末端异戊二烯化对于GSIS是必需的。我们还获得了初步的证据，表明有缺陷的表达的PPT酶，以及葡萄糖介导的激活特定的G-蛋白在胰岛从啮齿动物模型的2型糖尿病。基于这些和其他的初步数据，我们假设葡萄糖介导的PPT酶的激活是必要的GSIS从胰岛和缺陷的GSIS在体外和体内模型中的葡萄糖毒性和2型糖尿病的葡萄糖的PPT酶的葡萄糖激活的结果缺陷。我们将利用各种生物化学、生理学和分子生物学方法在正常大鼠胰岛和INS-1细胞中检验我们的假设。具体目的1是证明葡萄糖介导的PPT酶活化是分离的细胞中GSIS所必需的。我们将确定葡萄糖对分离的细胞中PPT酶的磷酸化状态和相关催化活化的调节。我们将通过转染显性阴性PPT酶突变体或通过siRNA介导的内源性PPT酶耗竭进一步确定GSIS中PPT酶激活的基本性质。具体目标2是证明葡萄糖介导的PPT酶活化的特定缺陷和G蛋白异戊烯化的相关异常在受损GSIS的体外和体内模型中导致胰岛素分泌缺陷。首先，我们将研究长期暴露于高血糖条件下的INS-1细胞或正常大鼠胰岛中PPTase信号通路的功能状态、候选G蛋白的异戊烯化和随后的GSIS。为了进一步验证我们的假设，我们将这些体外研究扩展到来自2型糖尿病啮齿动物模型的胰岛，即GK大鼠和ZDF大鼠。在本文中，我们将确定这些动物模型中GSIS的异常是否是由于葡萄糖诱导的PPT酶活化和特定G蛋白的异戊烯化的缺陷[例如，Rac 1]在这些小岛上。总之，我们认为，拟议的研究是第一个确定潜在的原因与影响的异常PPTase信号和GSIS在体外和体内模型受损GSIS。我们的目标是研究蛋白质异戊二烯化的生理控制在细胞刺激分泌耦合导致GSIS，并确定潜在的缺陷，在这个信号级联导致异常GSIS在模型中的葡萄糖毒性和糖尿病。

DESCRIPTION (provided by applicant): Emerging evidence implicates small G-proteins [e.g., Rac1 and Cdc42] in glucose-stimulated insulin secretion [GSIS] in the islet ¿ cell. However, the precise cellular mechanisms underlying glucose-mediated activation of these proteins remain only partially understood. Using generic [e.g., statins], and more specific pro-drug [e.g., allyl or vinyl farnesols and geranylgeraniols] and peptidomimetic [e.g., GGTI-2147] inhibitors of protein prenyltransferases [PPTases], we have recently reported that C-terminal prenylation of islet G- proteins is essential for GSIS. We have also obtained preliminary evidence to indicate defective expression of PPTases, as well as glucose-mediated activation of specific G-proteins in islets from rodent models of type 2 diabetes. Based on these and other preliminary data, we hypothesize that glucose-mediated activation of PPTases is necessary for GSIS from the islet and that deficient activation of PPTases by glucose results in defective GSIS in in vitro and in vivo models of glucotoxicity and type 2 diabetes. We will test our hypotheses in normal rat islets and INS-1 cells by utilizing various biochemical, physiological, and molecular biological approaches. Specific Aim 1 is to demonstrate that glucose-mediated activation of PPTases is necessary for GSIS in isolated ¿ cells. We will determine the regulation, by glucose, of the phosphorylation status and associated catalytic activation of PPTases in isolated ¿ cells. We will further determine the essential nature of PPTase activation in GSIS either via transfection of dominant negative PPTase mutants or via siRNA-mediated depletion of endogenous PPTases. Specific Aim 2 is to demonstrate that specific defects in glucose-mediated activation of PPTases, and associated abnormalities in G-protein prenylation culminates in insulin secretory defects in in vitro and in vivo models of impaired GSIS. First, we will examine the functional status of the PPTase signaling pathway, prenylation of candidate G-proteins and subsequent GSIS in INS-1 cells or normal rat islets chronically exposed to hyperglycemic conditions. To further validate our hypothesis, we will extend these in vitro studies to islets derived from two rodent models of type 2 diabetes, namely the GK rat and the ZDF rat. Herein, we will determine if abnormalities in GSIS in these animal models are due to defects in glucose-induced activation of PPTases and prenylation of specific G-proteins [e.g., Rac1] in these islets. Together, we believe that the proposed studies are the first to determine potential cause vs. effect for the abnormalities in PPTase signaling and GSIS in in vitro and in vivo models of impaired GSIS. Our goals are to examine the physiological control of protein prenylation in ¿ cell stimulus-secretion coupling leading to GSIS, and to determine potential defects in this signaling cascade leading to abnormalities in GSIS in models of glucotoxicity and diabetes.