Regulation of Intestinal Bile Acid Transport

描述(申请人提供)：大部分胆汁酸通过心尖钠依赖型胆汁酸转运体(ASBT)在肠道吸收。HASBT功能的紊乱已被证明会改变血浆胆固醇水平，并与各种胆固醇相关疾病的生理机制有关。然而，到目前为止，ASBT在人体肠道中调节的分子机制还不是很清楚。为了确定胆汁酸吸收与胆固醇相关疾病之间的分子联系，关键是要阐明正常情况下以及糖尿病激素失衡情况下hASBT的调节机制(S)。在这方面，我们实验室的大量初步研究利用人肠道Caco 2细胞作为体外细胞模型，证明了短期和长期暴露于胰岛素和胰升糖素后hASBT功能和表达的调节。这项研究旨在广泛研究Caco-2细胞中胰岛素和胰升糖素对hASBT表达的影响，以及信号转导途径、膜转运事件。在具体目标1中，我们将利用蛋白激酶的特异性抑制剂、siRNA和免疫沉淀技术，重点描述短期暴露于胰岛素和胰高血糖素时参与ASBT调控的信号通路。在特定目标2中提出的研究将检查hASBT在质膜不同亚区(脂筏)中的分布，以及膜转运事件在胰岛素和胰高血糖素对其调节中的作用。脂筏将通过Optiprep密度梯度上溶解的质膜浮选来分离。对于膜转运的研究，融合到ASBT的绿色荧光蛋白将被转染到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将侧重于阐明胰岛素和高血糖素引起ASBT表达改变的分子机制。启动子分析和凝胶漂移分析将为胰岛素和胰升糖素对ASBT转录调控提供重要信息。鉴于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，并加强我们对胆汁酸吸收与脂质和胆固醇动态平衡之间联系的分子基础的理解。更好地了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法，以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.