Oversupply and underutilization of lipid fuels are widely recognized to be strongly associated with insulin resistance in skeletal muscle. Recent attention has focused on the mechanisms underlying this effect, and defects in mitochondrial function have emerged as a potential player in this scheme. Because evidence indicates that lipid oversupply can produce abnormalities in extracellular matrix composition and matrix changes can affect the function of mitochondria, the present study was undertaken to determine whether muscle from insulin-resistant, nondiabetic obese subjects and patients with type 2 diabetes mellitus had increased collagen content. Compared with lean control subjects, obese and type 2 diabetic subjects had reduced muscle glucose uptake (P Ͻ 0.01) and decreased insulin stimulation of tyrosine phosphorylation of insulin receptor substrate-1 and its ability to associate with phosphatidylinositol 3-kinase (P Ͻ 0.01 and P Ͻ 0.05). Because it was assayed by total hydroxyproline content, collagen abundance was increased in muscle from not only type 2 diabetic patients but also nondiabetic obese subjects (0.26 Ϯ 0.05, 0.57 Ϯ 0.18, and 0.67 Ϯ 0.20 g/mg muscle wet wt, lean controls, obese nondiabetics, and type 2 diabetics, respectively), indicating that hyperglycemia itself could not be responsible for this effect. Immunofluorescence staining of muscle biopsies indicated that there was increased abundance of types I and III collagen. We conclude that changes in the composition of the extracellular matrix are a general characteristic of insulin-resistant muscle. insulin resistance; extracellular matrix; type 2 diabetes mellitus INTENSIVE INVESTIGATION into the biochemical and molecular mechanisms underlying insulin resistance has yielded detailed knowledge of insulin signaling in the classical target tissues of insulin, skeletal muscle, fat, and liver. A variety of abnormalities has been revealed in skeletal muscle, including decreased GLUT4 translocation, glycogen synthase and hexokinase activity, and decreased insulin receptor and insulin receptor substrate (IRS)-1 tyrosine phosphorylation and activation of phosphatidylinositol (PI) 3-kinase. Many of the defects in insulin action in muscle can be attributed to decreased insulin signaling (5, 17). However, the causes underlying insulinsignaling defects are unknown. Evidence has accumulated that indicates that mitochondrial abnormalities also exist in insulinresistant skeletal muscle. There are structural changes in muscle mitochondria in insulin resistance (14), decreased expression of nuclear-encoded mitochondrial genes (10,19,21), and evidence of decreased mitochondrial function in vivo (22,23). A key abnormality is the decreased ability of skeletal muscle mitochondria to oxidize fat in insulin-resistant individuals (15). The combination of these abnormalities potentially leads to accumulation of intracellular fatty acid metabolites, including ceramides (1) and fatty acyl-CoAs (37), both of which can produce insulin-signaling abnormalities, possibly by...