Project 3: Longitudinal Effects of Air Pollution on Obesity in Mice (Allayee)

最近人类流行病学证据将空气污染与肥胖和代谢增加联系起来 在使用小鼠模型来识别潜在的疾病方面已经引起了相当大的兴趣。 生物机制。在这方面，有限的研究表明，暴露于高浓度的环境 颗粒物质（PM）在致肥胖饮食的情况下增加肥胖和胰岛素抵抗。 此外，这些研究集中在PM2.5（空气动力学直径S 2.5 pm），并使用 只有一个暴露期的横断面研究设计。相比之下，涉及中枢神经系统的研究 系统表型采用纳米级PM（nPM;空气动力学直径2 200 nm）， 进入大脑发挥作用虽然PM2.5和nPM都能可靠地诱导 组织中的氧化应激和炎症，nPM具有与组织中的氧化应激和炎症相对应的陡峭的近道路梯度。 儿童健康研究（CHS）中近道路空气污染（NRAP）与肥胖之间的关系 在项目1中描述。尽管存在这些关联，但代谢和/或炎症变化的顺序 导致肥胖的原因尚不清楚。因此，了解这些病理生理机制可能会 对保护人口免受当前和未来最严重的空气污染影响的重要意义 关心以解决这些关键障碍。项目3将进行综合试验， C57 BL/6小鼠肥胖模型，并将联合收割机在出生时减少产仔数（以诱导营养过剩 在早期生活中）在断奶时用高脂肪喂养。使用纵向研究设计，将小鼠 在产前暴露于nPM（暴露组）或过滤空气（对照组）的新型近道路源， 出生后或出生前和出生后发育。将对小鼠进行肥胖相关代谢， 5周龄（青春期）、9周龄时的分子、生物化学和神经生物学表型 (late青春期）和13周龄（成年早期）。在具体目标1中，我们将确定身体 通过磁共振成像评估葡萄糖/胰岛素组成（瘦组织质量和全身脂肪）， 通过腹膜内葡萄糖耐量试验（IPGTT）进行代谢，测量血浆中的一组脂肪细胞因子， 并测定肝脏脂质含量。在具体目标2中，我们将通过以下方式表征小鼠脂肪组织： 免疫组织化学以确定冠状结构（CLS）的存在，这表明 巨噬细胞浸润和炎症，并通过流式细胞术定量巨噬细胞亚型（M1/M2）。 将进行外植体孵育研究，以确定WFRO中脂肪细胞因子的产生，并实时检测 PCR将用于研究分离的脂肪细胞中的炎症和代谢基因表达， 巨噬细胞在具体目标3中，我们将确定nPM暴露对肥胖的影响是否 通过控制代谢调节的下丘脑中的神经生物学途径介导。 免疫组织化学技术和形态学分析将被用来表征组织， 参与摄食调节的下丘脑神经投射。代谢相关的表达 神经肽基因也将通过实时PCR和食物摄入以核特异性方式进行研究 将进行评估，以确定nPM暴露是否会导致摄食行为改变。 综上所述，拟议的研究提供了几个层次的创新：（1）与中心一致 重点关注NRAP，我们将使用在主要交通走廊附近收集的nPM，我们之前已经证明， 在体外和体内具有可证实的生化和分子效应;（2）nPM反映了近道 在生物学相关的纳米级尺寸部分中的梯度，其在元素碳中增强， （3）新的收集和暴露程序将保留 原始气溶胶和已知的这种nPM转移到体循环中并进入 器官，包括大脑;（4）肥胖小鼠模型反映了人类肥胖的自然生命过程 通过将生命早期的营养过剩与断奶时的高脂肪喂养相结合;以及5）在断奶期间将动物暴露于nPM， 三个发育阶段将确定敏感性的关键窗口。结果。项目3高度 与该中心的其他项目相结合，并将通过阐明 NRAP对肥胖和代谢失调的影响的病理生理机制。的 我们将在小鼠中获得的生理表型，如全身组成，葡萄糖耐量， 肝脏脂肪沉积，将与研究CHS参与者的项目1获得的结果相当 从终生暴露于空气污染的极端情况中选出。在以下水平获得的小鼠表型 包括组织学、脂肪细胞因子释放和细胞特异性基因表达， 与项目2中建议的从CHS受试者中获得的结果相当。因此，实验 在项目3中提出的将产生有意义的洞察的时间顺序和方向性的影响， nPM在小鼠肥胖的三个重要方面，并可能提供因果关系的信息，可以指导 项目1和2中的知情分析，因为在人类中收集了类似的肥胖相关参数。

Recent epidemiological evidence in humans linking air pollution with increased adiposity and metabolic diseases has garnered considerable interest in the use of mouse models to identify potential underlying biological mechanisms. In this regard, limited studies have shown that exposure to concentrated ambient particulate matter (PM) increases adiposity and insulin resistance in the context of an obesogenic diet. Furthermore, these studies have focused on PM2.5 (aerodynamic diameter S 2.5pm) and been done using cross-sectional study designs with only one exposure period. By comparison, studies involving central nervous system phenotypes have employed nanoscale PM (nPM; aerodynamic diameter 2 200nm), which have been shown to enter the brain where they have functional effects. Although both PM2.5 and nPM reliably induce oxidative stress and inflammation in tissues, nPM have steep near-roadway gradients corresponding to the associations between near-roadway air pollution (NRAP) and obesity in the Children's Health Study (CHS) described in Project 1. Despite these associations, the sequence of metabolic and/or inflammatory changes that lead to obesity are not known. Understanding these pathophysiological mechanisms could thus have important implications for protecting the population from air pollution exposures of greatest current and future concern. To address these critical barriers. Project 3 will carry out comprehensive experiments with the wellestablished C57BL/6 mouse model of obesity and will combine litter reduction at birth (to induce over-nutrition dunng early life) with high fat feeding at the time of weaning. Using a longitudinal study design, mice will be exposed to a novel near-roadway source of nPM (exposed group) or filtered air (control group) dunng prenatal, postnatal, or both pre and postnatal development. Mice will be characterized for obesity-related metabolic, molecular, biochemical, and neurobiological phenotypes at 5 weeks of age (puberty period), 9 weeks of age (late adolescence), and 13 weeks of age (young adulthood). In Specific Aim 1, we will determine body composition (lean tissue mass and whole body fat) by magnetic resonance imaging, assess glucose/insulin metabolism by intraperitoneal glucose tolerance tests (IPGTTs), measure a panel of adipocytokines in plasma, and determine hepatic lipid content. In Specific Aim 2, we will characterize adipose tissue from mice by immunohistochemistry to determine the presence of crown-like structures (CLS), which is indicative of macrophage infiltration and inflammation, and by flow cytometry to quantitate macrophage subtypes (M1/M2). Explant incubation studies will be carried out to determine in wfro production of adipocytokines and real-time PCR will be used to investigate inflammatory and metabolic gene expression in both isolated adipocytes and macrophages. In Specific Aim 3, we will determine whether the effects of nPM exposure on obesity are mediated through neurobiological pathways in the hypothalamus that control metabolic regulation. Immunohistochemical techniques and morphometric analyses will be used to characterize the organization of hypothalamic neural projections involved in feeding regulation. Expression of metabolically-relevant neuropeptide genes will also be investigated in a nucleus-specific manner by real-time PCR and food intake will be assessed to determine whether nPM exposure results in altered feeding behavior. Taken together, the proposed studies offer several levels of innovation: (1) consistent with the Center focus on NRAP, we will use nPM collected near a major traffic corridor and which we have previously shown to have demonstrable biochemical and molecular effects in vitro and in vivo; (2) the nPM reflects the nearroadway gradient in the biologically relevant nanoscale size fraction that is ennched in elemental carbon and metals of vehicular source; (3) the novel collection and exposure procedure will preserve the size distnbution of the original aerosol and the known potential of such nPM to translocate into the systemic circulation and into organs, including the brain; (4) the mouse model of obesity reflects the natural life course of obesity in humans by coupling over-nutrition in early life with high fat feeding at weaning; and 5) exposing animals to nPM during three developmental stages will identify critical window(s) of susceptibility. As a result. Project 3 is highly integrated with the other projects of this Center and will complement the human studies by elucidating the pathophysiological mechanisms that underlie the effects of NRAP on obesity and metabolic dysregulation. The physiological phenotypes that we will obtain in mice, such as whole body composition, glucose tolerance, and hepatic fat deposition, will be comparable to those obtained by Project 1, which will study CHS participants selected from the extremes of lifetime exposure to air pollution. The murine phenotypes obtained at the levels of adipose tissue, including histology, adipocytokine release, and cell-specific gene expression, will similarty be equivalent to those being obtained from CHS subjects as proposed in Project 2. Thus, the experiments proposed in Project 3 will yield meaningful insight into the temporal sequence and directionality of the effects of nPM on three important aspects of obesity in mice and may provide causal information that could guide informed analyses in Projects 1 and 2, as analogous obesity-related parameters are collected in humans.