Blood-brain barrier regulates leptin transport in obesity

描述（由申请人提供）： 瘦素与血脑屏障 (BBB) 的相互作用在某些形式的肥胖中发挥着重要作用。 在本提案中，我们将阐明瘦素如何调节一些与进食相关的肽和细胞因子穿过血脑屏障的渗透的新方面。 (a) 为了检验瘦素招募选择性饱腹感肽受体并激活运输的假设，我们将研究瘦素如何增加另一种有效的饱腹感肽尿皮质素的血液到大脑的渗透。 我们将首先确定瘦素和尿皮质素受体是否参与该过程。 然后，在小鼠 TM-BBB4 脑内皮细胞上共转染表达受体后，我们将通过免疫共沉淀和配体结合测定来确定这些受体是否存在异二聚化。 此外，我们将通过免疫荧光显微镜确定这些受体在与瘦素结合后是否共定位于内吞囊泡上。 (b) 为了检验瘦素可以增强现有细胞因子转运系统从而减少进食的假设，我们将研究瘦素如何上调肿瘤坏死因子 α (TNFα) 的转运。 我们预测，瘦素将通过调节 p55 受体、p75 受体和与 TNFα 转运相关的蛋白质的磷酸化来增加 TNFα 流入，如转运测定、免疫沉淀和蛋白质印迹所示。 (c)为了测试瘦素介导的TNFα和尿皮质素转运调节的生理相关性，我们将测量正常喂养的小鼠和食物匮乏的小鼠中TNFα、尿皮质素以及瘦素本身的转运。 我们预测，瘦素只会在自由进食的小鼠中增加尿皮质素和 TNFα 的转运，从而在循环中瘦素已经相对较高时向大脑提供额外的饱腹感信号。 相比之下，我们预测，在食物匮乏的小鼠中，瘦素和 TNFα 的转运系统都将下调，而尿皮质素的转运系统不会被激活，正如在没有食物的严重条件下对食欲抑制剂的预期一样。 通过完成拟议的研究，我们将证明 BBB 上存在新型蛋白质-蛋白质相互作用、这些相互作用所涉及的机制，以及 BBB 在调节进食方面的额外功能作用。 这些信息不仅有助于更好地理解 BBB 转运的一般机制，而且有助于摄入肽的潜在治疗用途。

DESCRIPTION (provided by applicant): The interactions of leptin with the blood-brain barrier (BBB) play an important role in some forms of obesity. In this proposal, we will elucidate the novel aspects of how leptin regulates the permeation of some feeding-related peptides and cytokines across the BBB. (a) to test the hypothesis that leptin recruits receptors of selective satiety peptides and activates transport, we will examine how leptin increases the blood-to-brain permeation of urocortin, another potent satiety peptide. We will first determine whether receptors for both leptin and urocortin are involved in this process. We will then determine whether there is heterodimerization of these receptors by use of co-immunoprecipitation and ligand binding assays, after the receptors are expressed by co-transfection on mouse TM-BBB4 brain endothelial cells. Further, we will determine by immunofluorescent microscopy whether there is co-localization of these receptors on endocytotic vesicles after binding to leptin. (b) To test the hypothesis that leptin can enhance an existing transport system for a cytokine that also reduces feeding, we will examine how leptin upregulates the transport of tumor necrosis factor alpha (TNFalpha). We predict that leptin will crease TNFalpha influx by modulating phosphorylation of the p55-receptors, p75-receptors, and proteins related to TNFalpha transport, as shown by transport assays, immunoprecipitation, and Western blot. (c) To test the physiological relevance of leptin-mediated regulation of TNFalpha and urocortin transport, we will measure the transport of TNFalpha, urocortin, as well as leptin itself in mice that are fed normally and in mice that are food-deprived. We predict that leptin will increase urocortin and TNFalpha transport only in mice with free access to food, thereby providing additional satiety signals to the brain when leptin is already relatively high in the circulation. In contrast, we predict that in food-deprived mice, the transport systems for both leptin and TNFalpha will be down-regulated and that for urocortin will not be activated, as expected for anorectic agents under the severe condition in which no food is available. By completing the proposed studies, we will demonstrate the presence of novel protein-protein interactions at the BBB, the mechanisms involved in these interactions, and the additional functional role of the BBB in regulating feeding. This information will not only add to a better understanding of the mechanisms of BBB transport in general but also to the potential therapeutic use of ingestive peptides.