Consequences of astrocytic leptin receptor upregulation on obesity

描述(申请人提供)：这项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多症。我们观察到，星形胶质细胞中的OBR基因敲除导致小鼠对饮食诱导的肥胖产生部分抵抗，这一发现与在神经元中OBR基因敲除的小鼠中发现的肥胖相反。此外，成年肥胖小鼠表现出星形胶质细胞增生和下丘脑选择性核团星形细胞OBR的强烈上调。这些OBR是有功能的，因为瘦素治疗激活了原代星形胶质细胞中的多个信号通路。因此，我们假设OBR(+)星形胶质细胞通过两种主要方式提供促进肥胖的负性调节：(A)通过加速瘦素的更新来减少神经元对瘦素的可获得性；(B)通过改变释放的胶质递质的轮廓来调节神经元的功能。星形胶质细胞的这种作用提供了一种将神经炎症过程和体重控制联系起来的直接机制。我们将在三个具体目标上检验这些假设。(1)为了证明反应性星形胶质细胞促进瘦素的周转并减弱神经元中的瘦素信号，Aim 1将使用已建立的反应性星形胶质细胞增生性肥胖模型来确定侧脑室注射瘦素后荧光结合的瘦素和pSTAT3信号的分布。结果将与星形胶质细胞特异性瘦素受体基因敲除小鼠的结果进行比较，并在星形胶质细胞抑制剂氟柠檬酸预处理后进行比较。(2)在目标2中，我们将检验瘦素调节神经胶质递质产生的假设，包括急性期的谷氨酸和三磷酸腺苷，以及稍后的细胞因子。原代星形胶质细胞和神经元将用于钙成像，并将测试星形胶质细胞条件培养液和神经元-神经胶质混合培养的效果。(3)目的3将确定星形胶质细胞特异性瘦素受体基因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相关联。总体而言，这些结果将证明OBR(+)星形胶质细胞在调节神经元瘦素信号方面发挥着重要作用。由于星形胶质细胞在神经损伤中起关键作用，也是神经免疫轴的组成部分，这些研究将提供神经炎症调节体重的切实机制。 公共卫生意义：瘦素是一种主要由脂肪组织产生的荷尔蒙。高瘦素血症可见于肥胖和代谢综合征。肥胖及其相关的高脂血症、心血管并发症、癌症和睡眠呼吸暂停的发病率在全国和全球范围内都在急剧上升。这项研究将阐述星形胶质细胞如何参与将瘦素输送到神经元并调节其行为。星形胶质细胞是大脑中含量最丰富的细胞，显然与脑损伤有关，但很少有研究表明它们是否与瘦素和肥胖有关。我们最近发现瘦素受体的mRNA和蛋白确实存在于星形胶质细胞中。此外，在成年肥胖症小鼠模型中，这些瘦素受体的表达水平增加。这表明星形细胞瘦素系统在肥胖者的调节变化中起着重要作用。用小鼠和培养的细胞进行了一系列实验，旨在研究这些OBR(+)星形胶质细胞如何影响神经元功能和整体代谢表型。因此，它们的相关性在于：(A)更好地了解星形胶质细胞如何影响肥胖的发生和发展；(B)更好地了解大脑中细胞与细胞的相互作用；以及(C)潜在地确定新的治疗靶点，以更好地对抗肥胖症。

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.