PROJECT SUMMARY/ABSTRACT Overnutrition is central to the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD). This research proposal addresses the unanswered question of how molecular mechanisms that normally promote energy conservation become maladaptive and promote MASLD in response to overnutrition. The long-term goal of this research is to understand the regulatory relationships between cellular lipid molecules and metabolism. The objective of this research is to understand fundamental mechanisms for the regulation of energy homeostasis and nutrient metabolism. Our central hypothesis is that Them1 conserves energy by limiting thermogenesis in brown and beige adipose tissue through its functions both as a lipid- regulated enzyme that reduces rates of fatty acid oxidation, and as a transcriptional co-regulator. In obesity, we postulate that Them1 becomes maladaptive. In addition to limiting energy expenditure in thermogenic adipose tissue, high fat diet-induced Them1 upregulation in liver leads to steatosis and excess gluconeogenesis. The rationale for the proposed research is that the mechanisms by which Them1 limits energy expenditure, while promoting hepatic steatosis and insulin resistance, will reveal specific new targets for the management of MASLD. Guided by extensive preliminary data, the central hypothesis will be tested in three specific aims: 1) To establish the mechanisms by which Them1 regulates triglyceride metabolism; 2) To elucidate transcriptional regulation of nutrient homeostasis by Them1; and 3) To delineate lipid-mediated regulation of Them1 cellular and enzymatic activities. In Aim 1, we will utilize biochemical and biophysical approaches to test the hypothesis that dynamic formation of membrane free biomolecular condensates (puncta) and their dissolution in brown adipose tissue provides a tissue-specific physiological mechanism for rapid suppression of thermogenesis. We will define whether static puncta are responsible for Them1-mediated regulation of hepatic lipid droplets. Aim 2 will test the hypothesis that enzymatically active Them1 within the nucleus regulates gene transcription. Using mouse models and cell culture systems, we will leverage recombinant adeno-associated virus to express wild type and Them1 mutant constructs that probe the influence of cellular localization and enzymatic activity on gene transcription profiles. We will define nuclear interacting partners for Them1 in liver and brown adipose tissue that contribute to the regulation of nutrient homeostasis. Aim 3 will test the hypothesis that lipid molecules bind and regulate the cellular localization and enzymatic activity of Them1. We will determine whether bound lysophosphatidylcholines and fatty acids control the exposure and functional utility of a nuclear localization signal located within the Them1 START domain. We will elucidate the allosteric control of Them1 activity by lipid binding. Overall, this proposal will el...