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 fro m animal studies and clinical trials with non-insulin-dependent diabet es mellitus patients that Troglitazone might reduce insulin resistance . The molecular mechanism of this effect is not understood. In this st udy, we investigated whether Troglitazone might interfere with the mec hanism of glucose-induced insulin resistance. Several studies indicate that hyperglycemia reduces the kinase activity of the insulin recepto r 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 desensit ization is mediated by activation of PKC. We studied the effect of hyp erglycemia 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 w ere acutely exposed to hyperglycemic conditions in the absence or pres ence of Troglitazone (0.01-2 mu g/ml). The insulin receptor was solubi lized from a plasma membrane fraction or whole cell lysates, and prote ins were separated by sodium dodecyl sulfate-polyacrylamide gel electr ophoresis and immunoblotted against antiphosphotyrosine and anti-insul in receptor p-subunit (CT 104) antibodies. Acute hyperglycemia (25 mM glucose) induced a significant inhibition of the insulin receptor kina se (IRK) activity within 30 min (inhibition to 30 +/- 12.5% of maximal insulin-stimulated P-subunit phosphorylation, n = 9, P < 0.01). The g lucose-induced inhibition of the insulin receptor kinase could be anta gonized by a preincubation of the cells with Troglitazone before addit ion of 25 mM glucose (72 +/- 13.5% of maximal insulin-stimulated P-sub unit phosphorylation after 20-30 min of preincubation at a concentrati on of 2 mu g/ml Troglitazone, n = 9, P < 0.01). In addition, Troglitaz one is also able to reverse the inhibition of the insulin receptor kin ase caused by a prior glucose incubation. In parallel with the decreas e of the IRK activity, the phosphorylation of the insulin receptor sub strate-1 (IRS-1) was inhibited (inhibition to 45 +/- 11.8% of maximal insulin-stimulated IRS-1 phosphorylation, n = 4, P < 0.01), and this i nhibition also could be reduced by Troglitazone (84 +/- 15.7% of maxim al insulin-stimulated IRS-1 phosphorylation in the presence of 2 mu g/ ml Troglitazone, n = 4, P < 0.01). Taken together, the data suggest th at Troglitazone is able to prevent and reverse hyperglycemia-induced i nsulin resistance of the insulin receptor in rat-1 fibroblasts. This m echanism might be relevant for the in vivo activity of this compound.