PROJECT SUMMARY Insulin resistance is commonly associated with obesity and is a major contributor to the development of obesity-related metabolic diseases, (e.g. nonalcoholic fatty liver disease [NAFLD] and type 2 diabetes). Most when characterized by glucose tolerance and insulin sensitivity. However, a subset of people with obesit adverse metabolic effects of excess adiposity. A better understanding of the mechanisms responsible for the differences in metabolic function between people with MHO and MUO can provide important mechanistic insights into the metabolic heterogeneity of obesity and possibly identify novel targets for therapeutic intervention. Small extracellular vesicles (sEVs, or exosomes) are produced by cells throughout the body and deliver bioactive molecules via the bloodstream to regulate metabolic processes in multiple organ systems. Studies in mice have found that sEVs released from adipose tissue modulate insulin sensitivity and contain regulatory miRNAs and bioactive lipids that alter insulin signaling. Consistent with these findings, we recently found that plasma and adipose tissue sEVs obtained from people with obesity and NAFLD impair insulin signaling in cultured myotubes, suggesting sEVs also contribute to the regulation of insulin sensitivity in people. In this proposal, we will evaluate the metabolic effects and cargo content of sEVs isolated from plasma and adipose tissue (AT) explant cultures obtained from three groups of participants. We will characterize participants based on adiposity and metabolic health: 1) metabolically healthy lean (MHL, BMI 18.5-24.9 kg/m2 and normal insulin sensitivity, defined as homeostatic model assessment of insulin resistance (HOMA-IR) <2); 2) metabolically healthy obese (MHO, BMI 30.0-44.9 kg/m2 and HOMA-IR<2); and 3) metabolically unhealthy obesity (MUO, BMI 30.0-44.9 kg/m2 and HOMA-IR>2.5). We posit that: i) sEVs from plasma and adipose tissue in people who are MUO have adverse effects on insulin action compared with sEVs from people who are MHL or MHO; ii) these metabolic differences are associated with differences in sEV content of miRNA and bioactive lipids; and iii) moderate 10% weight loss in people with MUO changes the miRNA and lipid content of plasma and adipose tissue sEVs and their effects on insulin action. The following specific aims will be addressed: 1) determine the effects of sEVs obtained from plasma and adipose tissue of MHL, MHO and MUO participants on: i) insulin action, gene expression and protein profile in cells vitro and in mice in vivo; 2) identify differences in miRNA and bioactive lipid profiles of plasma and adipose tissue sEVs obtained from MHL, MHO and MUO groups; and 3) determine whether a 10% weight loss-induced increase in insulin sensitivity in the MUO group will alter plasma and adipose tissue sEVs miRNA and lipid content and ameliorate the adverse effects of plasma and adipose tissue sEVs on insulin action in in vitro and in mice in vivo. These...