The worldwide prevalence of obesity and type 2 diabetes continue to increase substantially in the United States and globally. Regional fat distribution and ectopic adipose deposition are paramount when considering obesity- associated metabolic dysfunction, as we now know that adipose tissue is an important endocrine organ, secreting a variety of bioactive factors that impact nearby tissues. One such ectopic adipose depot, intermuscular adipose tissue (IMAT), accumulates within the skeletal muscle compartment, between muscle fibers. While previous research has shown that IMAT directly relates to insulin resistance and type 2 diabetes, mechanistic studies aimed at addressing how IMAT is involved in the development of metabolic disease are lacking. Mounting evidence suggests that extracellular matrix (ECM) protein deposition and remodeling is associated with insulin resistance in skeletal muscle, and recent studies in our lab indicate that IMAT may secrete a greater amount of ECM proteins compared to other adipose depots such as visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT). However, IMAT-derived ECM components have never been investigated in relation to skeletal muscle insulin sensitivity. Therefore, the goal of this proposal is to identify novel mechanisms by which IMAT impacts the development of type 2 diabetes, focusing on the central hypothesis that IMAT secretes ECM proteins that promote skeletal muscle insulin resistance. The first aim of this proposal will test this hypothesis by expanding upon preliminary data and evaluating the degree to which IMAT, VAT and SAT secrete extracellular matrix proteins that relate to insulin sensitivity in humans. Conditioned media using human IMAT, SAT and VAT tissue biopsies will be generated, and secreted Fibronectin (FN), Collagen VI (ColVI), Fibrinogen-beta (FGB) and Transforming growth factor beta-1 (TGFB1) will be measured using a combination of multiplex ELISA and proteomic analyses. The second aim of this proposal will determine the ability of these specific extracellular matrix proteins to increase skeletal muscle insulin resistance in vitro. IMAT, SAT, and VAT conditioned media will be administered to differentiated L6 myotube cultures to measure changes in insulin sensitivity. In addition, the ability of FN, ColVI, FGB, and TGFB1 to individually modulate insulin sensitivity will be measured by isolated treatment of recombinant proteins as well as administration of FN, ColVI, FGB and TGFB1-null conditioned media. To summarize, these studies will explore potentially novel mechanisms regulating skeletal muscle insulin sensitivity, highlighting the essential role of IMAT in the development of insulin resistance and type 2 diabetes.