Zone-specific mitochondrial functions in regulation of hepatic metabolism

.SUMMARY-R21-纽加德 衰老和营养过剩对肝脏健康有有害影响，包括线粒体的发育。 功能障碍。肝脏被分成不同的区域(区域1-区域3)，每个区域都富含 代谢途径，但对衰老和代谢应激如何影响区带特异性线粒体知之甚少 代谢或呼吸功能。使用一套强大的技术，包括为区域设计的鼠标- 免疫隔离标签(Mito-tag)的特异性表达，可使线粒体得到纯化 分钟，先进的代谢体和代谢流量分析工具，线粒体呼吸分析，以及 在活体CRISPR基因发现技术中，我们将(I)优化我们的工作流程，以使用Mito-Tag进行快速 以高产量和完全代谢功能从肝脏中分离线粒体(目标1)；ii)开发区- 特定Mito-Tag小鼠允许全面观察分离的线粒体的代谢和能量学 来自不同肝区(目标2)；(3)部署两种体内CRISPR筛查方法，包括一种新的 本实验室设计的方法，以确定影响肝细胞适应能力的线粒体基因 营养应激(目标3)。通过准确地了解新陈代谢是如何在初级肝脏中跨区域划分的 细胞，并通过我们的CRISPR筛查识别增强每个区域肝细胞福祉的基因， 我们可以为迅速流行的肝功能障碍的新疗法的开发做出贡献。 美国人口老龄化。

.SUMMARY – R21 – Newgard . Aging and overnutrition have deleterious effects on liver health, including development of mitochondrial dysfunction. The liver is divided into distinct zones (Zone 1–Zone 3), each enriched in a discrete subset of metabolic pathways, but little is known about how aging and metabolic stress affect zone-specific mitochondrial metabolic or respiratory functions. Using a powerful suite of technologies, including mice engineered for zone- specific expression of an immunoisolation tag (MITO-Tag) that allows purification of mitochondria in <10 minutes, sophisticated metabolomic and metabolic flux analysis tools, mitochondrial respiratory assays, and in vivo CRISPR gene discovery technologies, we will (i) optimize our workflow for use of MITO-Tag for rapid isolation of mitochondria from liver at high yield and with full metabolic functionality (Aim 1); ii) develop zone- specific MITO-Tag mice to allow a comprehensive view of metabolism and energetics in mitochondria isolated from different liver zones (Aim 2); (iii) deploy two in vivo CRISPR screening approaches, including a novel method designed in our laboratory, to identify mitochondrial genes that impact hepatocellular fitness during nutritional stress (Aim 3). By understanding exactly how metabolism is partitioned across zones in primary liver cells, and by identifying genes via our CRISPR screens that enhance the well-being of liver cells in each zone, we can contribute to development of novel therapies for the growing pandemic of liver dysfunction in the rapidly aging US population.