Troglitazone Prevents Glucose-Induced Insulin Resistance of Insulin Receptor in Rat-1 Fibroblasts

Troglitazone (CS045), a compound belonging to the thiazolidine diones, is being tested as a new oral antidiabetic agent. Evidence exists from animal studies and clinical trials with non-insulin-dependent diabetes mellitus patients that Troglitazone might reduce insulin resistance. The molecular mechanism of this effect is not understood. In this study, we investigated whether Troglitazone might interfere with the mechanism of glucose-induced insulin resistance. Several studies indicate that hyperglycemia reduces the kinase activity of the insulin receptor in different cell types. This effect is paralleled by translocation of several protein kinase C (PKC) isoforms, and it can be prevented by PKC inhibitors, which suggests that glucose-induced receptor desensitization is mediated by activation of PKC. We studied the effect of hyperglycemia on the insulin receptor kinase activity and its modulation by Troglitazone in rat-1 fibroblasts that stably overexpress the human insulin receptor. Before stimulation with insulin (10⁻⁷ M), cells were acutely exposed to hyperglycemic conditions in the absence or presence of Troglitazone (0.01–2 μ/ml). The insulin receptor was solubilized from a plasma membrane fraction or whole cell lysates, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted against antiphosphotyrosine and anti-insulin receptor β-subunit (CT 104) antibodies. Acute hyperglycemia (25 mM glucose) induced a significant inhibition of the insulin receptor kinase (IRK) activity within 30 min (inhibition to 30 ± 12.5% of maximal insulin-stimulated β-subunit phosphorylation, n = 9, P < 0.01).The glucose-induced inhibition of the insulin receptor kinase could be antagonized by a preincubation of the cells with Troglitazone before addition of 25 mM glucose (72 ± 13.5% of maximal insulin-stimulated 3-subunit phosphorylation after 20–30 min of preincubation at a concentration of 2 μg/ml Troglitazone, n = 9, P < 0.01). In addition, Troglitazone is also able to reverse the inhibition of the insulin receptor kinase caused by a prior glucose incubation. In parallel with the decrease of the IRK activity, the phosphorylation of the insulin receptor substrate-1 (IRS-1) was inhibited (inhibition to 45 ± 11.8% of maximal insulin-stimulated IRS-1 phosphorylation, n = 4, P ± 0.01), and this inhibition also could be reduced by Troglitazone (84 ± 15.7% of maximal insulin-stimulated IRS-1 phosphorylation in the presence of 2 μg/ml Troglitazone, n = 4, P ± 0.01). Taken together, the data suggest that Troglitazone is able to prevent and reverse hyperglycemia-induced insulin resistance of the insulin receptor in rat-1 fibroblasts. This mechanism might be relevant for the in vivo activity of this compound.