Lipid droplets and the compartmentalization of subcellular metabolism - Lipid droplets (LDs), the organelles responsible for lipid storage and the largest energy reserve in most cell types, are the defining characteristic and etiological factor in the development of non-alcoholic fatty liver disease (NAFLD). Moreover, LDs are recognized to play central roles in coupling NAFLD to more systemic comorbidities such as Type 2 Diabetes and cardiovascular disease among others. LDs interact with numerous organelles, especially ER and mitochondria, which are thought to coordinate de novo LD biogenesis and fatty acid (FA) transfer/oxidation, respectively. However, published work from our laboratory and others have questioned the established dogma that direct transfer of FAs from LDs to mitochondria is the primary route of their oxidation during fasting. Using a multifaceted approach involving organelle proteomics, isotope tracing, and numerous super resolution microscopy approaches, we show for the first time that in the liver, the proteomes and metabolism of mitochondria attached to LDs (peridroplet mitochondria, PDM) support lipid anabolic pathways, whereas mitochondria unattached to LDs (cytosolic mitochondria, CM) a geared for enhanced FA oxidation and OXPHOS. Moreover, our data point to an important role for mitochondrial-associated membranes (MAMs), an ER domain that tightly interacts with mitochondria, as a key component in regulating LD-mitochondria interactions and dynamics. Collectively, these data suggest that interactions with LDs profoundly affect organelle dynamics and function. Based upon these data, the objective of this application is to define how LD interactions affect lipid metabolism and sensing to coordinate ER and mitochondrial function under physiological and pathophysiological conditions. We hypothesize that interactions of LDs with ER and mitochondria are critical modulators of MAM lipid sensing and mitochondrial function that govern hepatic lipid and energy metabolism. To test this hypothesis, we propose the following three specific aims: Aim 1 - To comprehensively define LD- mitochondria interactions and their impact on FA trafficking; Aim 2 - To determine the mechanisms through which subpopulations of MAM differentially impact mitochondrial bioenergetics and lipid metabolism; and Aim 3 - To determine how NAFLD impacts LD/MAM/mitochondria dynamics in NAFLD. To complete these aims, we will employ a wide range of advanced super resolution imaging approaches, proteomics and RNA sequencing, isotope tracing, and other cell biology and biochemical approaches in cells, mouse models and human liver biopsies. Upon completion of these studies, we will expect that we will have revealed novel mechanisms through which LDs can alter cellular function/dysfunction that underlie NAFLD etiology. We anticipate that this work will open new areas of research into intracellular signaling dynamics, which will advance therapeutic approaches targeting NAFLD and related comorbidities.
