We recently described the identification of a non-peptidyl fungal metabolite (L-783,281, compound 1), which induced activation of human insulin receptor (IR) tyrosine kinase and mediated insulin-like effects in cells, as well as decreased blood glucose levels in murine models of Type 2 diabetes (Zhang, B., Salituro, G., Szalkowski, D., Li, Z., Zhang, Y., Royo, I., Vilella, D., Diez, M. T., Pelaez, F., Ruby, C., Kendall, R. L., Mao, X., Griffin, P., Calaycay, J., Zierath, J. R., Heck, J. V., Smith, R. G. & Moller, D. E. (1999) Science 284, 974 -977). Here we report the characterization of an active analog (compound 2) with enhanced IR kinase activation potency and selectivity over related receptors (insulin-like growth factor I receptor, epidermal growth factor receptor, and platelet-derived growth factor receptor). The IR activators stimulated tyrosine kinase activity of partially purified native IR and recombinant IR tyrosine kinase domain. Administration of the IR activators to mice was associated with increased IR tyrosine kinase activity in liver. In vivo oral treatment with compound 2 resulted in significant glucose lowering in several rodent models of diabetes. In db/db mice, oral administration of compound 2 elicited significant correction of hyperglycemia. In a streptozotocin-induced diabetic mouse model, compound 2 potentiated the glucose-lowering effect of insulin. In normal rats, compound 2 improved oral glucose tolerance with significant reduction in insulin release following glucose challenge. A structurally related inactive analog (compound 3) was not effective on insulin receptor activation or glucose lowering in db/db mice. Thus, small molecule IR activators exert insulin mimetic and sensitizing effects in cells and in animal models of diabetes. These results have implications for the future development of new therapies for diabetes mellitus.Insulin elicits a diverse array of biological responses by binding to its specific receptor (1). The insulin receptor (IR) 1 is a heterotetrameric protein consisting of two extracellular ␣ subunits and two transmembrane  subunits. The binding of the ligand to the ␣ subunit of IR not only concentrates insulin at its site of action, but also induces conformational changes in the receptor, which in turn stimulates the tyrosine kinase activity intrinsic to the  subunit of the IR. Extensive studies have indicated that the ability of the receptor to autophosphorylate and phosphorylate intracellular substrates is essential for its mediation of the complex cellular responses of insulin (2-5).Insulin receptors trans-phosphorylate several immediate substrates (on Tyr residues), including insulin receptor substrate (IRS) proteins 1-4, Shc, and Gab 1, each of which provide specific docking sites for other signaling proteins containing Src homology 2 domains (6). These events lead to the activation of downstream signaling molecules, including phosphatidylinositol 3-kinase (PI 3-kinase). Numerous studies have adduced that PI 3-kinase is required for the metabolic effec...