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，rac1和cd42]与葡萄糖刺激的胰岛细胞的胰岛素分泌[GSIS]有关。然而，葡萄糖介导的这些蛋白质激活的确切细胞机制仍然只有部分了解。利用非专利药物[如他汀类药物]和更具体的前体药物[如烯丙基或乙烯基法呢醇和香叶香叶醇]和多肽类[如GGTI-2147]蛋白戊烯基转移酶[PPTase]抑制剂，我们最近报道了胰岛G蛋白的C末端预烯基化在GSIS中是必不可少的。我们还获得了初步证据，表明2型糖尿病啮齿动物模型的胰岛存在PPTase的缺陷表达，以及葡萄糖介导的特定G蛋白的激活。基于这些和其他初步数据，我们假设葡萄糖介导的PPTase的激活是胰岛GSIS所必需的，葡萄糖激活PPTase的缺陷在体外和体内都会导致糖毒性和2型糖尿病的GSIS模型。我们将利用各种生化、生理和分子生物学方法在正常大鼠胰岛和INS-1细胞中测试我们的假设。具体目的1是证明在分离的细胞中，葡萄糖介导的PPTase激活是GSIS所必需的。我们将确定葡萄糖对分离的细胞中PPTase的磷酸化状态和相关的催化激活的调节。我们将通过导入显性阴性PPTase突变体或通过siRNA介导的内源性PPTase耗尽来进一步确定GSIS中PPTase激活的本质。具体目的2是证明在体外和体内GSIS受损模型中，葡萄糖介导的PPTase激活的特异性缺陷以及相关的G蛋白预烯基化异常最终导致胰岛素分泌缺陷。首先，我们将研究长期暴露在高血糖条件下的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蛋白的预烯化以及随后的GSIS。为了进一步验证我们的假设，我们将把这些体外研究扩展到来自两种2型糖尿病啮齿动物模型的胰岛，即GK大鼠和ZDF大鼠。在这里，我们将确定这些动物模型中GSIS的异常是否由于葡萄糖诱导的PPTase激活和这些胰岛中特定G蛋白[例如，rac1]的预烯化缺陷所致。总之，我们相信所提出的研究是第一次确定在体外和体内GSIS受损模型中PPTase信号和GSIS异常的潜在原因和效果。我们的目标是研究细胞刺激-分泌偶联中蛋白质预烯基化对导致GSIS的生理控制，并确定在糖毒性和糖尿病模型中这一信号级联中导致GSIS异常的潜在缺陷。

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.