Consequences of astrocytic leptin receptor upregulation on obesity

描述（由申请人提供）：拟议的研究旨在确定星形细胞瘦素受体（ObR或LR）如何影响肥胖调节。肥胖和神经损伤一样，会导致区域性星形胶质细胞增生。我们观察到，敲除星形胶质细胞中的ObR导致小鼠对饮食诱导的肥胖产生部分抵抗，这与敲除神经元中ObR的小鼠的肥胖发现相反。此外，患有成年型肥胖的小鼠同时表现出星形胶质细胞增生和下丘脑选择性核中星形胶质细胞ObR的强烈上调。这些ObR是功能性的，因为瘦素治疗激活了原代星形胶质细胞中的多种信号通路。因此，我们假设ObR（+）星形胶质细胞通过两种主要方式提供负调控以促进肥胖：(a)通过加速瘦素的转换来减少瘦素对神经元的可用性；(b)通过改变释放的胶质递质谱来调节神经元功能。星形胶质细胞的这种作用为神经炎症过程和体重控制提供了直接机制。我们将在三个具体目标中检验这些假设。(1)为了证明反应性星形胶质细胞促进瘦素的转换并减弱大脑中神经元的瘦素信号，Aim 1将使用一个已建立的反应性星形胶质细胞形成模型——成人发病的肥胖——来确定瘦素在脑室内递送后荧光偶联的瘦素和pSTAT3信号的分布。结果将与星形胶质细胞特异性瘦素受体敲除小鼠的结果进行比较，并与星形胶质细胞抑制剂氟柠檬酸盐预处理后的结果进行比较。(2)在Aim 2中，我们将验证瘦素在急性期调节胶质递质（包括谷氨酸和ATP）和后期细胞因子的产生谱的假设。我们将使用原代星形胶质细胞和神经元进行钙成像，并测试星形胶质细胞条件培养基和混合神经元-胶质细胞培养的效果。(3)目的3将确定星形胶质细胞特异性瘦素受体敲除对代谢表型的功能影响，并将其与神经炎症的血清生物标志物联系起来。总之，这些结果将证明ObR（+）星形胶质细胞在调节神经元瘦素信号传导中的重要作用。由于星形胶质细胞在神经损伤中起着至关重要的作用，是神经免疫轴的组成部分，这些研究将为神经炎症调节体重提供切实的机制。

DESCRIPTION (provided by applicant): The proposed study aims to determine how astrocytic leptin receptors (ObR, or LR) affect obesity regulation. Obesity, like neural injury, results in regional astrogliosis. We observed that knockout of ObR in astrocytes leads to partial resistance of the mice to diet-induced obesity, a finding opposite to the obesity found in mice with knockout of ObR in neurons. Moreover, mice with adult-onset obesity show both astrogliosis and robust upregulation of astrocytic ObR in selective nuclei of the hypothalamus. These ObR are functional, as leptin treatment activates multiple signaling pathways in primary astrocytes. We, therefore, hypothesize that the ObR (+) astrocytes provide negative regulation to facilitate obesity in two main ways: (a) reducing availability of leptin to neurons by accelerating leptin turnover; (b) modulating neuronal function by alterations of the profile of glial transmitters released. Such a role for astrocytes provides a direct mechanism linking neuroinflammatory processes and body weight control. We will test the hypotheses in three specific aims. (1) To show that reactive astrocytes facilitate the turnover of leptin and attenuate neuronal leptin signaling in the brain, Aim 1 will use an established model of reactive astrogliosis - adult-onset obesity - to determine the distribution of fluorescently conjugated leptin and pSTAT3 signaling after intracerebroventricular delivery of leptin. The results will be compared with those from astrocyte specific leptin receptor knockout mice, and after pretreatment with the astrocyte inhibitor fluorocitrate. (2) In Aim 2, we will test the hypothesis that leptin modulates the production profile of gliotransmitters, including glutamate and ATP in the acute phase and cytokines at a later time. Primary astrocytes and neurons will be used for calcium imaging, and the effects of astrocyte conditioned medium and mixed neuron-glial culture will be tested. (3) Aim 3 will identify functional consequences of astrocyte specific leptin receptor knockout on the metabolic phenotype and correlate this with serum biomarkers of neuroinflammation. Overall, the results will demonstrate an essential role of ObR(+) astrocytes in the regulation of neuronal leptin signaling. As astrocytes are crucially involved in neural injury and an integral part of the neuroimmune axis, these studies will provide tangible mechanisms by which neuroinflammation can regulate body weight. PUBLIC HEALTH RELEVANCE: Leptin is a hormone mainly produced by fat tissue. Hyperleptinemia is seen in obesity and the metabolic syndrome. The incidence of obesity and its associated hyperlipidemia, cardiovascular complications, cancer, and sleep apnea have been on a steep rise nationally and globally. This study will address how astrocytes participate in delivering leptin to neurons and modulating its actions. Astrocytes are the most abundant cells in the brain, clearly involved in brain injury, but very few studies have addressed whether they are involved with leptin and obesity. We recently found that both the mRNA and protein of leptin receptors are indeed present in astrocytes. Moreover, the expression level of these leptin receptors increases in mouse models of adult-onset obesity. This suggests an important role of the astrocytic leptin system in the regulatory changes in obese subjects. A series of experiments with mice and cultured cells are designed to address how these ObR(+) astrocytes affect neuronal function and overall metabolic phenotype. Thus, their relevance lies in (a) better understanding of how astrocytes affect obesity onset and progression; (b) better understanding of cell-cell interactions in the brain; and (c) potential identification of novel therapeutic targets to better combat obesity.