Hypothalamic Integration of Energy Homeostasis Signals

对肥胖的稳态控制取决于大脑感知和响应身体能量储存循环指标变化的能力。该建议的重点是了解下丘脑弓状核中两组神经元的分子和细胞机制，这些神经元以Agouti相关的表达为标志， 蛋白质（Agrp）和前阿黑皮素原（Pomc），测量并响应瘦素、胰岛素和生长激素释放肽的变化。我们的方法已经成为可能的转基因技术和分子成像的最新进展，是基于工具的发展，使我们能够控制基因表达，特别是在Agrp/Npy和Pomc/Cart神经元。 利用细菌人工染色体（BAC）克隆在大肠杆菌中的同源重组，大肠杆菌，然后将修饰的克隆转基因导入小鼠中，我们将Cre重组酶置于Agrp或Pomc基因的调控元件的控制下。这些Tg.AgrpCre和Tg.PomcCre小鼠将被应用于更好地理解外周血细胞如何在体内生长。 通过构建和分析在Agrp神经元、Pomc神经元或两者中缺乏Stat 3或Irs 2的小鼠的表型，身体燃料储存和膳食模式的信号被下丘脑神经元感知和整合。我们还将使用一种新开发的测定方法，动态测量单个细胞中磷酸肌醇3-激酶信号，以确定弓状核神经元的每个亚群对瘦素、胰岛素或生长激素释放肽的反应 在脑切片制备中。最后，我们将确定增加长链脂酰辅酶A水平的药物是否以及如何与Agrp和Pomc神经元中的Stag和PI 3 K信号交叉。总之，这些研究将为控制下丘脑关键神经元对激素和营养相关信号的反应的细胞机制提供基本的新见解。

Homeostatic control of adiposity depends on the ability of the brain to sense and respond to changes in circulating indicators of body energy stores. This proposal is focused on understanding the molecular and cellular mechanisms by which two groups of neurons in the arcuate nucleus of the hypothalamus, those marked by expression of Agouti-related protein (Agrp) and Preproopiomelanocortin (Pomc), measure and respond to changes in leptin, insulin, and ghrelin. Our approach has been made possible by recent advances in transgenic technology and molecular imaging, and is based on the development of tools that allow us to control gene expression specifically in the Agrp/Npy and Pomc/Cart neurons. Using homologous recombination of bacterial artificial chromosome (BAC) clones in E. coli followed by transgenic introduction of the modified clones into mice, we have placed Cre recombinase under control of regulatory elements for the Agrp or Pomc genes. These Tg.AgrpCre and Tg.PomcCre mice will be applied to better understand how peripheral signals of body fuel stores and meal patterning are perceived and integrated by hypothalamic neurons by constructing and analyzing the phenotype of mice that are deficient for Stat3 or for Irs2 in either Agrp neurons, Pomc neurons, or both. We will also use a newly developed assay for dynamic measurement of phosphoinositide 3-kinase signaling in individual cells to determine how each subpopulation of arcuate nucleus neurons responds to leptin, insulin, or ghrelin in a brain slice preparation. Finally, we will determine if and how agents that increase long chain fatty acyl CoA levels intersect with Stag and PI3K signaling in Agrp and Pomc neurons. Together, these studies will provide fundamental new insight into cellular mechanisms governing the response of key hypothalamic neurons to hormonal and nutrient-related signals.