Small molecule insulin receptor activators potentiate insulin action in insulin-resistant cells.

In type 2 diabetes, impaired insulin signaling leads to hyperglycemia and other metabolic abnormalities. To study a new class of antidiabetic agents, we compared two small, nonpeptide molecules that activate insulin receptor (IR) beta-subunit tyrosine kinase activity: Merck L7, a direct IR agonist, and Telik's TLK16998, an IR sensitizer. In rat hepatoma cells (HTCs) that overexpress the IR (HTC-IR), IR autophosphorylation was directly activated by L7 in the absence of insulin. TLK16998 did not directly activate IR autophosphorylation, but it enhanced IR autophosphorylation in the presence of insulin. Tyrosine phosphorylation of an endogenous 185-kDa IR substrate was also significantly enhanced by both Merck L7 alone and TLK16998 plus insulin. Adding TLK16998 to L7 produced synergistic effects, further indicating that these two compounds act on the IR through separate mechanisms. We next studied HTC-IR(Delta485-599) cells, which overexpress a mutant IR with a deletion in the alpha-subunit connecting domain that does not undergo autophosphorylation in response to insulin binding. L7 was able to directly activate autophosphorylation of the deletion mutant IR in these cells, whereas TLK16998 had no effect. Compounds were then tested in three other cell models of impaired IR function. Both TLK16998 and Merck L7 improved IR autophosphorylation in cells with diminished IR signaling due to either treatment with tumor necrosis factor-alpha or overexpression of membrane glycoprotein PC-1. However, in TPA (tetradecanoylphorbol acetate)-treated cells, TLK16998 but not Merck L7 was able to significantly reverse the impaired insulin-stimulated IR autophosphorylation. In summary, these two classes of IR activators selectively increased IR function in a variety of insulin-resistant cell lines.

A novel small molecule that directly sensitizes the insulin receptor in vitro and in vivo.

Insulin resistance, an important feature of type 2 diabetes, is manifested as attenuated insulin receptor (IR) signaling in response to insulin binding. A drug that promotes the initiation of IR signaling by enhancing IR autophosphorylation should, therefore, be useful for treating type 2 diabetes. This report describes the effect of a small molecule IR sensitizer, TLK16998, on IR signaling. This compound activated the tyrosine kinase domain of the IR beta-subunit at concentrations of 1 micromol/l or less but had no effect on insulin binding to the IR alpha-subunit even at much higher concentrations. TLK16998 alone had no effect on IR signaling in mouse 3T3-L1 adipocytes but, at concentrations as low as 3.2 micromol/l, enhanced the effects of insulin on the phosphorylation of the IR beta-subunit and IR substrate 1, and on the amount of phosphatidylinositol 3-kinase that coimmunoprecipitated with IRS-1. Phosphopeptide mapping revealed that the effect of TLK16998 on the IR was associated with increased tyrosine phosphorylation of the activation loop of the beta-subunit tyrosine kinase domain. TLK16998 also increased the potency of insulin in stimulating 2-deoxy-D-glucose uptake in 3T3-L1 adipocytes, with a detectable effect at 8 micromol/l and a 10-fold increase at 40 micromol/l. In contrast, only small effects were observed on IGF-1-stimulated 2-deoxy-D-glucose uptake. In diabetic mice, TLK16998, at a dose of 10 mg/kg, lowered blood glucose levels for up to 6 h. These results suggest, therefore, that small nonpeptide molecules that directly sensitize the IR may be useful for treating type 2 diabetes.