Obesity is the most common and powerful force for creating insulin resistance and metabolic syndrome, however, the molecular basis of this association is not well understood. In this proposal, three independently funded researchers—Philip Kern, MD a clinical investigator, and Charlotte Peterson, PhD and Robert McGehee, PhD, with significant experience in muscle and adipocyte biology, respectively—will formalize a collaborative effort as a natural extension of previous work and shared interests in the fields of obesity, insulin resistance, and tissue lipid accumulation. Our overall hypothesis is that insulin resistance in humans stems largely from ectopic accumulation of intramyocellular lipid (IMCL) during the development of obesity. Further, we hypothesize that excess IMCL accumulation is dependent on secretory proteins derived from a complex interplay between adipocytes and macrophages in adipose tissue. To test these hypotheses, we will examine the interactions among adipocytes, macrophages, and muscle cells isolated and cultured from subjects that are obese with insulin resistance and impaired glucose tolerance (IGT), and from some with Type 2 Diabetes. This study population has elevated IMCL and is at high risk for obesity complications, but avoids the pathophysiologic complications of glucotoxicity. These subjects will be compared to obese subjects with normal glucose tolerance (NGT).