Direct effect of cholesterol on glucose-stimulated insulin secretion

描述(由申请人提供)： 这项拟议研究的总体目标是调查胆固醇，包括血清和胰岛胆固醇，在表征2型糖尿病进展的胰腺细胞功能障碍中的作用。胰腺细胞功能的改变导致胰岛素对葡萄糖的分泌反应受损，这是从糖尿病前期向糖尿病状态转变的标志。在许多致病因素中，脂毒性是指循环中游离脂肪酸(FFAs)或细胞脂肪含量升高的糖尿病效应，已被广泛研究。尽管肥胖患者的胆固醇经常伴随着游离脂肪酸的升高，但胆固醇在胰腺细胞葡萄糖刺激的胰岛素分泌(GSIS)中的作用还没有很好的文献报道。与使用高脂饮食喂养的小鼠的研究不同，在高脂饮食小鼠的研究中，血浆胆固醇的上升伴随着血浆游离脂肪酸的增加，我们建立了隔离的胰岛系统，其中胆固醇水平在体内使用apoE缺陷的小鼠，在体外使用药物试剂，而不改变细胞的游离脂肪酸水平。我的初步数据显示，血清胆固醇升高、胰岛胆固醇升高和GSIS降低之间存在直接联系，而正常分泌可以通过消耗胆固醇来恢复。我还发现，在GSIS中起关键作用的两种蛋白质，神经元型一氧化氮合酶(NNOS)和葡萄糖激酶(GK)，其功能依赖于胰岛素颗粒上富含胆固醇的膜微区。通过这项拟议的研究，我希望从我的初步数据中巩固结论，并为胆固醇依赖的GSIS提供详细的分子机制。具体目标是：1)从胰腺细胞中确定胆固醇(饮食、血浆和胰岛)在GSIS中的作用。2)明确胆固醇对胰腺细胞GK调节的影响。3)探讨膜微域在nNOS对胰腺细胞GK调节中的作用。这些研究将为更全面地了解肥胖诱导的2型糖尿病提供洞察力，并表明存在一种新的机制，将高脂血症与2型糖尿病的发病机制联系起来，这种机制独立于游离脂肪酸。 相关性：(-细胞功能障碍导致GSIS降低被认为是2型糖尿病最早和最相关的体征。关于高脂血症和GSIS之间的联系的研究主要集中在游离脂肪酸上。这项研究中提出的胆固醇对细胞代谢的直接影响将开启一套新的机制，可能有助于肥胖患者的细胞功能障碍和糖尿病的发病。

DESCRIPTION (provided by applicant): The overall goal of the proposed study is to investigate the role of cholesterol, including serum and islet cholesterol, in pancreatic (-cell dysfunction that characterizes the progression of type 2 diabetes. Alteration of pancreatic (-cell function leading to an impaired insulin secretory response to glucose is a hallmark of the transition from the prediabetic to the diabetic state. Among the many contributing factors, lipotoxicity, which refers to the diabetogenic effect of elevated circulating free fatty acids (FFAs) or cellular fat content, has been studied extensively. Despite the fact that cholesterol is often elevated in obese patients along with FFAs, the role of cholesterol in glucose-stimulated insulin secretion (GSIS) from pancreatic (-cells is not well documented. Unlike studies using mice fed on high-fat diets in which a rise in plasma cholesterol is accompanied by increased plasma FFAs, we established systems of isolated islets where cholesterol levels were altered, both in vivo using apoE-deficient mice and in vitro using pharmacological reagents, without changing cellular FFA levels. My preliminary data show a direct link between elevated serum cholesterol, elevated islet cholesterol and reduced GSIS, and that normal secretion can be restored by cholesterol depletion. I also show that the function of two proteins critically involved in GSIS, neuronal nitric oxide synthase (nNOS) and glucokinase (GK), depends on cholesterol-enriched membrane microdomains on the insulin granules. Through performing the proposed study I hope to solidify the conclusions from my preliminary data and provide a detailed molecular mechanism for cholesterol-dependent GSIS. The specific aims are: 1) Determine the role of cholesterol (dietary, plasma, and islet) in GSIS from pancreatic (-cells. 2) Identify the effect of cholesterol on GK regulation in pancreatic (-cells. 3) Investigate the role of membrane microdomains in nNOS regulation of GK in pancreatic (-cells. These studies will provide insights to a more complete understanding of obesity-induced type 2 diabetes and indicate the existence of a novel mechanism linking hyperlipidemia and pathogenesis of type 2 diabetes that is independent of FFAs. Relevance: (-cell dysfunction leading to decreased GSIS is considered the earliest and most relevant sign of type 2 diabetes mellitus. Studies on the link between hyperlipidemia and GSIS have focused mostly on FFAs. The direct effect of cholesterol on (-cell metabolism proposed in this study would open a novel set of mechanisms that may contribute to (-cell dysfunction and the onset of diabetes in obese patients.