Activation of PKC theta is associated with lipid-induced insulin resistance and PKC theta knockout mice are protected from the lipid-induced defects. However, the exact mechanism by which PKC theta contributes to insulin resistance is not known. To investigate whether an increase in PKC theta expression leads to insulin resistance, C2C12 skeletal Muscle cells were transfected with PKC theta DNA and treated with different concentrations of insulin for 10 min. PKC theta overexpression induced reduction of IRS-1 protein levels with a decrease in insulin-induced p85 binding to IRS-1, phosphorylation of PKB and its substrates, p70 and GSK3. Pretreatment of these cells with GF-109203X (a nonspecific PKC inhibitor, IC50 for PKC theta = 10 nM) recovered insulin signaling. PKC theta was found to be expressed in liver and treatment of human hepatoma cells (HepG2) with high insulin and glucose resulted in ail increase in PKC theta expression that correlated with a decrease in IRS-1 protein levels and the development of insulin resistance. Reduction of PKC theta expression using RNAi technology significantly inhibited the degradation of IRS-1 and enhanced insulin-induced IRS-1 tyrosine phosphorylation, p85 association to IRS-1 and PKB phosphorylation. In conclusion, by overexpressing PKC theta or using RNAi technology to downregulate PKC theta. we have demonstrated that PKC theta has a key role in the development of insulin resistance. These findings suggest that PKC theta mediates not only insulin resistance in muscle but also in liver, which may contribute to the development of whole body insulin resistance and diabetes. (c) 2006 Elsevier Inc. All rights reserved.