Regulation of Intestinal Bile Acid Transport

描述（由申请方提供）：大多数胆汁酸通过顶端钠依赖性胆汁酸转运蛋白（ASBT）在肠道中吸收。hASBT功能紊乱已被证明可改变血浆胆固醇水平，并与各种胆固醇相关疾病的病理生理学有关。然而，迄今为止，人类肠道中ASBT调节的分子机制还不清楚。为了确定胆汁酸吸收和胆固醇相关疾病之间的分子联系，关键是要阐明在正常条件下以及在糖尿病中所见的激素失衡条件下hASBT的调节机制。在这方面，来自我们实验室的广泛的初步研究已经证明了hASBT功能和表达的调节响应于短期和长期暴露于胰岛素和胰高血糖素，利用人肠Caco 2细胞作为体外细胞模型。拟定的研究旨在广泛研究Caco-2细胞中胰岛素和胰高血糖素应答的信号转导途径、膜运输事件以及hASBT表达的改变。在具体的目标1中，我们将重点描绘参与ASBT调节的信号通路，通过短期暴露于胰岛素和胰高血糖素，利用特异性蛋白激酶抑制剂，siRNA和免疫沉淀技术。具体目标2中提出的研究将检查hASBT质膜不同亚结构域（脂筏）的分布以及膜运输事件在胰岛素和胰高血糖素对其调节中的作用。将通过在Optiprep密度梯度上漂浮溶解的质膜来分离脂筏。对于膜运输研究，将与ASBT融合的绿色荧光蛋白转染到Caco 2细胞中，然后利用共聚焦显微镜进行活细胞成像。具体目标3将集中于阐明响应于胰岛素和胰高血糖素的ASBT表达改变的分子机制。启动子分析和凝胶迁移分析将产生重要的信息，胰岛素和胰高血糖素的ASBT的转录调控。考虑到这些内分泌激素在代谢过程整合中的核心作用，拟议的研究将提供有关hASBT调控的关键新数据，并增强我们对胆汁酸吸收与脂质和胆固醇稳态之间联系的分子基础的理解。更好地理解胆汁酸吸收在胆固醇相关疾病中的作用可能有助于推进新的治疗方式来对抗高胆固醇血症和动脉粥样硬化等疾病。

DESCRIPTION (provided by applicant): The majority of bile acids are absorbed in the intestine via Apical Sodium Dependent Bile Acid Transporter, ASBT. Disturbances in hASBT function have been shown to alter the level of plasma cholesterol and implicated in the phathophysiology of various cholesterol-related disorders. To date, however, the molecular mechanisms of ASBT regulation in the human intestine are not well understood. In order to define the molecular link between bile acid absorption and cholesterol-related disorders, it is critical to elucidate the mechanism(s) of regulation of hASBT under normal conditions as well as in conditions of hormonal imbalance as seen in diabetes mellitus. In this regard, extensive preliminary studies from our laboratory have demonstrated the modulation of hASBT function and expression in response to both short-term and long-term exposure to insulin and glucagon, utilizing human intestinal Caco2 cells as an in vitro cellular model. The proposed studies are designed to extensively investigate the signal transduction pathways, membrane trafficking events as well as alterations in the expression of hASBT in response to insulin and glucagon in Caco-2 cells. In specific aim 1, we will focus on delineating the signaling pathways involved in ASBT regulation by short-term exposure to insulin and glucagon by utilizing specific inhibitors of protein kinases, siRNA and immunoprecipitation techniques. Studies proposed in specific aim 2 will examine the distribution of hASBT indifferent subdomains (lipid rafts) of the plasma membrane and the role of membrane trafficking events in their regulation by insulin and glucagon. Lipid rafts will be isolated by floatation of solubilized plasma membrane on Optiprep density gradient. For membrane trafficking studies, green fluorescent protein fused to ASBT will be transfected in Caco2 cells followed by live cell imaging utilizing confocal microscopy. Specific aim 3 will focus on elucidating the molecular mechanisms underlying the altered expression of ASBT in response to insulin and glucagon. Promoter analysis and gel shift assays will yield important information on the transcriptional regulation of ASBT by insulin and glucagon. Given the central role for these endocrine hormones in the integration of metabolic processes, the proposed studies would provide critical novel data about the regulation of hASBT and enhance our understanding of the molecular basis for the link between bile acid absorption and the lipid and cholesterol homeostasis. A better understanding of the role of bile acid absorption in cholesterol related disorders may help advance new therapeutic modalities to counter diseases such as hypercholesterolemia and atherosclerosis.