Project Summary Excess adiposity – obesity – causes insulin resistance (IR) in white adipose tissue (WAT) and other tissues. The role of poor WAT quality in IR, however, is less clear. Low adipocyte triglyceride (TG) turnover, high formation rates of new adipocytes, high senescent cell burden, tissue and cellular inflammation and high circulating free fatty acids (FFAs) all characterize poor WAT quality. Exercise enhances whole-body IR and decreases fasting FFAs, independent of reductions in fat mass, thus highlighting the significance of exercise effects on WAT quality. Evidence of the direct impact of exercise – independent of weight loss – on IR within WAT is limited and inconclusive. The underlying mechanisms that drive exercise-induced changes in WAT biology, such as new adipocyte formation and TG turnover rates, cell composition, senescence, and inflammation, are poorly understood in humans. Ours will be the first study to comprehensively assess the direct effects of exercise on WAT-specific IR and WAT quality in vivo and ex vivo in humans. We have conducted groundbreaking studies of in vivo formation of new adipocytes and adipocyte TG turnover rates using a metabolic labeling protocol that incorporates deuterium (2H), administered as 2H2O, into the DNA and triglycerides of adipose cells. We now have compelling published and preliminary evidence that exercise reduces in vivo formation of new adipocytes in both mice and humans, suggesting an important exercise-induced mechanism of WAT remodeling. We reason that increased adipocyte TG turnover rates improve adipocyte-specific and overall WAT quality and thus abrogate the need for increased formation of new adipocytes. Using our established method, we will employ innovative single nuclei transcriptomics (snRNAseq) to discover the effects of exercise on senescence and inflammation within adipocytes and non-adipocytes of WAT to further understand how exercise impacts WAT quality and WAT- specific IR via in vivo microdialysis during insulin suppression. In this study, we will perform a randomized, controlled clinical trial to evaluate the impact of 12 weeks of endurance exercise on abdominal subcutaneous WAT adipocyte formation and TG turnover in vivo, cell composition and WAT-specific lipolytic rates under physiological insulin suppression in men and pre-menopausal women with obesity. We hypothesize that endurance exercise will increase adipocyte TG turnover rates thereby decreasing formation of new adipocytes, the size of existing adipocytes and reducing inflammation and senescent cell burden in adipocytes and non- adipocytes within WAT. These cell-specific changes will play a key role in exercise-induced improvements in WAT-specific insulin-suppressed lipolytic rates in vivo and reduced fasting FFA levels. By performing comprehensive metabolic and molecular phenotyping of WAT in humans in response to exercise, these pivotal studies will expand our understanding of WAT remodeling and how change...