Zone-specific mitochondrial functions in regulation of hepatic metabolism

．R21 -纽加德

.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.