Intermuscular adipose tissue (IMAT) is marbled within skeletal muscle and appears to play a key role in the obesity-induced risk of type 2 diabetes. What is not known is how IMAT promotes decreased muscle insulin sensitivity. There is a critical need to understand how IMAT contributes to the risk of obesity-induced diabetes, and how to intervene to minimize IMAT-induced muscle metabolic dysfunction. The overall objective for this project is to determine the impact of weight loss on IMAT secretion of fibronectin and inflammatory cytokines and eicosanoids. Our central hypothesis is that IMAT secretion of fibronectin promotes muscle insulin resistance, and IMAT secretion of inflammatory cytokines and eicosanoids causes muscle inflammation, both of which are diminished by weight loss. The rationale that underlies the proposed research is that clarifying the extent to which weight loss alters the IMAT secretome will inform development of interventions to modify IMAT and improve muscle insulin sensitivity in obese individuals. We propose two specific aims: Specific Aim 1. Evaluate the impact of weight loss on IMAT secretion of fibronectin and the role of fibronectin in the IMAT secretome to decrease insulin sensitivity in vitro. Preliminary data inform our working hypothesis that IMAT secretes fibronectin that decreases muscle insulin sensitivity and is attenuated after weight loss in humans. In vitro experiments will measure the extent to which IMAT fibronectin secretion explains IMAT-induced muscle insulin resistance. We will study individuals with obesity before and after a 12-week weight loss intervention. IMAT will be sampled using an ultrasound guided IMAT biopsy technique, insulin sensitivity measured using insulin clamps, and IMAT content measured using MRI. Specific Aim 2 – Determine the influence of weight loss on IMAT secretion of pro-inflammatory cytokines and eicosanoids and potency to cause inflammation and decrease insulin sensitivity in vitro. We hypothesize, again based on preliminary data that the IMAT secretome is less inflammatory after weight loss in obese individuals, with decreased potency to promote muscle inflammation and insulin resistance. Muscle inflammatory response and insulin sensitivity with IMAT secretome exposure will be compared before and after weight loss in vitro. The proposed research is innovative because it represents a new and substantive departure from the status quo by testing specific IMAT secreted paracrine signals rather than clinical associations with IMAT content. These contributions will be significant by identifying the first IMAT paracrine signals that impact muscle and revealing the plasticity of IMAT through weight loss, providing proof of concept that IMAT is a therapeutic target to combat muscle metabolic dysfunction.