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起着重要作用。 在这个提议中，我们将阐明瘦素如何调节一些摄食相关肽和细胞因子穿过血脑屏障的渗透的新方面。 (a)为了检验瘦素募集选择性饱腹感肽的受体并激活转运的假设，我们将研究瘦素如何增加另一种有效饱腹感肽尿皮质素的血脑渗透。 我们将首先确定是否瘦素和尿皮质素受体参与这一过程。 然后，我们将确定是否有异源二聚化的这些受体通过使用免疫共沉淀和配体结合试验后，通过共转染小鼠TM-BBB 4脑内皮细胞上的受体表达。 此外，我们将通过免疫荧光显微镜确定是否有这些受体的共定位后，结合瘦素的内吞囊泡。 (b)为了验证瘦素可以增强现有的细胞因子的运输系统，也减少进食的假设，我们将研究瘦素如何上调肿瘤坏死因子α（TNF α）的运输。 我们预测，瘦素将通过调节p55-受体，p75-受体和TNF α转运相关蛋白的磷酸化来增加TNF α的流入，如转运试验，免疫沉淀和Western印迹所示。 (c)为了测试瘦素介导的调节TNF α和尿皮质素转运的生理相关性，我们将测量正常喂养的小鼠和食物剥夺的小鼠中TNF α、尿皮质素以及瘦素本身的转运。 我们预测，瘦素将增加尿皮质素和肿瘤坏死因子α的运输，只有在小鼠自由获取食物，从而提供额外的饱足感信号时，瘦素已经相对较高的循环。 与此相反，我们预测，在食物剥夺的小鼠，瘦素和TNF α的运输系统将下调，尿皮质素将不会被激活，预期在严重的条件下，没有食物的厌食剂。 通过完成拟议的研究，我们将证明在BBB中存在新的蛋白质-蛋白质相互作用，这些相互作用中涉及的机制，以及BBB在调节摄食中的额外功能作用。 这些信息不仅有助于更好地理解BBB转运的机制，而且有助于消化肽的潜在治疗用途。

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.