Excessive hepatic glucose production through the gluconeogenesis pathway is partially responsible for the elevated glucose levels observed in patients with type 2 diabetes mellitus (T2DM). The forkhead transcription factor forkhead box O1 (Foxo1) plays a crucial role in mediating the effect of insulin on hepatic gluconeogenesis. Here, using a db/db mouse model, we demonstrate the effectiveness of Foxo1 inhibitor, an orally active small-molecule compound, as a therapeutic drug for treating T2DM. Using mass spectrometric affinity screening, we discovered a series of compounds that bind to Foxo1, identifying among them the compound, 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (AS1842856), which potently inhibits human Foxo1 transactivation and reduces glucose production through the inhibition of glucose-6 phosphatase and phosphoenolpyruvate carboxykinase mRNA levels in a rat hepatic cell line. Oral administration of AS1842856 to diabetic db/db mice led to a drastic decrease in fasting plasma glucose level via the inhibition of hepatic gluconeogenic genes, whereas administration to normal mice had no effect on the fasting plasma glucose level. Treatment with AS1842856 also suppressed an increase in plasma glucose level caused by pyruvate injection in both normal and db/db mice. Taken together, these findings indicate that the Foxo1 inhibitor represents a new class of drugs for use in treating T2DM.

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The first step in insulin action is ligand stimulation of the insulin receptor tyrosine kinase leading to the subsequent tyrosine phosphorylation of various endogenous substrates including insulin receptor substrates IRS-1 and IRS-2 (1). Tyrosine phosphorylated IRS-1 and IRS-2 serve as docking proteins for Src-homology 2 domain containing proteins including the p85 regulatory subunit of phosphatidylinositol (PI) 1 3-kinase.This binding results in the membrane localization and activation of the p110 catalytic subunit of PI 3-kinase, leading to the generation of phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate. These lipid products induce the activation of a number of signaling kinases including the Ser/Thr kinase Akt (2), which is phosphorylated and activated by the upstream phosphoinositide-dependent kinase 1 (3). Activation of Akt and its downstream signals were shown to stimulate the metabolic actions of insulin such as GLUT4 translocation and glucose transport (4, 5) leading to the hypothesis that this enzyme may play a critical role in mediating this biological response. A number of subsequent studies have supported this hypothesis; knock-out mice and humans lacking Akt2 exhibit a decrease in insulin responsiveness (6, 7), and knockdown of Akt2 in adipocytes by either siRNA or genetically (8, 9) suppressed the ability of insulin to stimulate glucose uptake. However, other data indicate that Akt activation is not required for insulin stimulation of glucose transport and instead indicate that distinct signal cascades stimulated by insulin may be responsible for eliciting this response (10, 11).Most recently a study indicated that neomycin treatment of 3T3-L1 adipocytes would allow insulin-stimulated glucose transport in the presence of wortmannin, an inhibitor of PI 3-kinase and Akt activation (12). Because neomycin binds and sequesters PI 4,5-P 2 (13), it was proposed that insulin-stimulated glucose transport may occur in part via the "masking" of PI 4,5-P 2 (12). To better understand the mechanism whereby neomycin reverses wortmannin inhibition of insulin-stimulated glucose transport, we first investigated downstream targets of Akt in insulin-treated 3T3-L1 adipocytes. In the presence of wortmannin, neomycin allowed the insulin-stimulated uptake of glucose as reported previously (12). However, it also allowed the insulin-stimulated increase in phosphorylation of several downstream targets of Akt including the direct substrate PRAS40 (14), two downstream targets of Akt, ribosomal protein S6 and GSK3␤ (15, 16), as well as allowing insulin-stimulated activation of Akt itself. In addition, neomycin was found incapable of preventing the inhibition of a structurally unrelated PI 3-kinase inhibitor, LY294002 (17). Finally, neomycin was found to inactivate in vitro the inhibitory actions of wortmannin. These results indicate that the effects of neomycin in adipocytes are not mediated via its ability to sequester PI 4,5-P 2 , but is instead caused by the ability of neomycin to inacti...