White Adipose Tissue Physiology, Mitochondrial Function and Adiponectin Metabolic dysregulation with all of it pathophysiological sequelae, including diabetes, cardiovascular disease and cancer, continues to be on the rise. We need to identify new areas that can be targeted for improvements in systemic metabolism. Over the last funding period, we have made significant progress towards defining key processes in the white adipocyte that lead to mitochondrial challenges and consequently, impaired adipose tissue function. These include challenges to the mitochondrial import machinery through overexpression of Amyloid Precursor Protein , loss of mitochondrial DNA and a dysregulation in iron metabolism through overexpression of mitoNEET and mitoferritin. We have applied many of these challenges to other cell types of relevance in adipose tissue, such as macrophages and adipocyte precursor cells. Here, we propose to integrate these challenges and observe how they feed into the Integrated Stress Response of mitochondria. We will analyze how endoplasmic reticulum (ER) stress and the mitochondrial stress pathways result in altered cellular physiology of the white adipocyte. We can carefully time and titrate these mitochondrial manipulations, then probe them either in the presence or absence of the integral mitochondrial stress sensor, DELE1. We will examine these phenomena in the following areas: A) at the cellular level in the mature adipocyte; B) in the microenvironment at the level of whole adipose tissue physiology; C) at the whole-body level, assessing the systemic effects of the Integrated Stress Response. Specifically, we propose to address the underlying mechanisms with the following hierarchical approaches: In Aim 1, we will measure and manipulate mitochondrial stress. In Aim 2, we will manipulate mitochondrial matrix iron levels and assess the impact on the Integrated Stress Response. In Aim 3, we will address the interplay between the mitochondrial and ER stress phenomena. In Aim 4, we will determine the role that the Integrated Stress Response has in activating the removal and packaging of sub-mitochondrial particles through exosomes. Combined, this proposal will provide significant novel insights into the downstream consequences of mitochondrial dysfunction, ER stress and the Integrated Stress Response in the white adipocyte in vivo. We are uniquely positioned to address these aims with mouse models that we have generated and characterized during the initial ten years of this grant. In adipocytes, these interventions will severely tip the balance of lipid storage, lipid oxidation and carbohydrate metabolism, thereby contributing directly to the development of obesity and insulin resistance. These experiments will also enable us to carefully dissect the effects of altered mitochondrial function on the production of the critical adipokine, adiponectin. These are the first comprehensive in vivo experiments assessing the role of the Integrated S...