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

最近的人类流行病学证据表明，空气污染与肥胖和代谢增加有关 疾病已经引起了人们对使用小鼠模型来识别潜在的潜在潜在疾病的极大兴趣 生物机制。在这方面，有限的研究表明，暴露在浓缩环境中 在肥胖饮食的背景下，颗粒物(PM)会增加肥胖和胰岛素抵抗。 此外，这些研究还集中在PM2.5(空气动力学直径S 2.5 PM)上，并使用 只有一个曝光期的横断面研究设计。相比之下，涉及中枢神经的研究 系统表型采用了纳米级PM(NPM；空气动力学直径2200 nm)，已被 显示它们进入大脑，在那里它们有功能影响。尽管PM2.5和NPM都可靠地导致 组织中的氧化应激和炎症，NPM具有陡峭的近道路梯度，对应于 儿童健康研究(CHS)中路边空气污染(NRAP)与肥胖的关系 尽管有这些关联，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 保护人口免受当前和未来最严重的空气污染影响的重要影响 担忧。以解决这些关键障碍。项目3将对已建成的学生进行综合实验 建立C57BL/6小鼠肥胖模型，并结合出生时的产仔减少(以诱导过度营养 在断奶时饲喂高脂肪饲料。使用纵向研究设计，小鼠将被 暴露于新的近道路来源的NPM(暴露组)或过滤空气(对照组)浸泡产前， 出生后，或出生前和出生后的发育。小鼠的特征将是肥胖相关的代谢， 5周龄(青春期)、9周龄的分子、生化和神经生物学表型 (青春期后期)和13周(青春期)。在具体目标1中，我们将确定身体 磁共振成像测定组成(瘦组织质量和全身脂肪)，评估葡萄糖/胰岛素 通过腹膜内葡萄糖耐量试验(IPGTTS)代谢，测量血浆中一组脂肪细胞因子， 并测定肝脂含量。在特定目标2中，我们将通过以下方式来表征小鼠的脂肪组织 免疫组织化学用于确定冠状结构(CLS)的存在，这表明 用流式细胞仪对巨噬细胞亚型(M1/M2)进行定量。 将进行外植体培养研究，以确定脂肪细胞因子的体外产生和实时 将使用聚合酶链式反应研究炎症和代谢基因在分离的脂肪细胞和 巨噬细胞。在具体目标3中，我们将确定接触NPM对肥胖的影响是否 通过控制新陈代谢调节的下丘脑神经生物学途径进行调节。 免疫组织化学技术和形态计量分析将被用来表征组织 下丘脑神经投射参与摄食调节。代谢相关基因的表达 神经肽基因也将通过实时定量聚合酶链式反应和食物摄入量以核特异的方式进行研究。 将接受评估，以确定接触NPM是否会导致喂养行为的改变。 综上所述，拟议的研究提供了几个层面的创新：(1)与中心一致 关注NRAP，我们将使用在一条主要交通走廊附近收集的NPM，我们之前已经展示过 在体外和体内具有明显的生化和分子效应；(2)NPM反映了近道路 与生物相关的纳米级分数的梯度，这种分数固定在元素碳和 车载来源的金属；(3)新的收集和曝光程序将保留 原始气溶胶和已知的这种NPM转移到体循环和进入 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 将早期的过度营养与断奶时的高脂肪喂养结合起来；5)使动物在 三个发育阶段将确定易感性的关键窗口(S)。结果。项目3高度重视 与该中心的其他项目相结合，并将通过阐明 NRAP对肥胖和代谢失调影响的病理生理机制。这个 我们将在小鼠身上获得的生理表型，如全身成分、葡萄糖耐量和 肝脏脂肪沉积，将与项目1获得的结果相当，该项目将研究CHS参与者 从终生暴露在空气污染中的极端情况中挑选。在不同水平上获得的小鼠表型 脂肪组织，包括组织学，脂肪细胞因子的释放和细胞特异性基因的表达，将是相似的 相当于项目2中提议的从CHS受试者那里获得的数据。因此，这些实验 项目3中提出的建议将对以下影响的时间顺序和方向性产生有意义的洞察 NPM对小鼠肥胖的三个重要方面的研究，并可能提供可能指导 项目1和2中的知情分析，因为类似的与肥胖有关的参数是在人类身上收集的。

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.