PKCh is a key player in the development of insulin resistance Deanna Haasch, Cathleen Berg, Jill E. Clampit, Terry Pederson, Leigh Frost, Paul Kroeger, Cristina M. Rondinone * Metabolic Diseases Research, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA Received 13 February 2006 Available online 9 March 2006 Abstract Activation of PKCh is associated with lipid-induced insulin resistance and PKCh knockout mice are protected from the lipid-induced defects. However, the exact mechanism by which PKCh contributes to insulin resistance is not known. To investigate whether an increase in PKCh expression leads to insulin resistance, C2C12 skeletal muscle cells were transfected with PKCh DNA and treated with different concentrations of insulin for 10 min. PKCh 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 non- specific PKC inhibitor, IC 50 for PKCh = 10 nM) recovered insulin signaling. PKCh was found to be expressed in liver and treatment of human hepatoma cells (HepG2) with high insulin and glucose resulted in an increase in PKCh expression that correlated with a decrease in IRS-1 protein levels and the development of insulin resistance. Reduction of PKCh 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 PKCh or using RNAi technology to downregulate PKCh, we have demonstrated that PKCh has a key role in the development of insulin resistance. These findings suggest that PKCh mediates not only insulin resistance in muscle but also in liver, which may contribute to the development of whole body insulin resistance and diabetes. Ó 2006 Elsevier Inc. All rights reserved. Keywords: PKCh; Insulin resistance; Hepatocytes; IRS-1; akt/PKB Insulin resistance in target tissues such as skeletal muscle is a major factor contributing to the development of type 2 diabetes. Activation of nPKCs, including PKCh, has been correlated with insulin resistance in a number of studies, especially in association with increased lipid availability [1–3]. PKCh is a serine/threonine kinase and a member of the novel subfamily of PKC isoforms (d, e, h, and l). PKCh consists of 707 amino acid residues (73 kDa protein) with highest similarity to PKCd (67%) [4,5]. PKCh is expressed predominantly in skeletal muscle, T-cells, and platelets, with less expression in cardiac muscle, placenta, and liver [6]. It is the main isoform present in skeletal muscle, a major target tissue for insulin. In addition, expression of PKCh is higher in insulin-resistant white muscle fibers in comparison to red muscle fibers [7]. Increased activation and translocation of PKCh to the cell membrane has been shown to be associated with lip- id-induced insulin resistance in animals [1–3,8]. It is theo- rized that after fatty acids get into the muscle cell, a product of the fatty acids, such as fatty acyl CoA or diac- ylglycerol, activates PKCh. This leads to a serine/threonine phosphorylation cascade and increased serine phosphory- lation of IRS-1 (and possibly IRS-2), which in turn leads to decreased tyrosine phosphorylation of IRS-1, decreased activity of PI3-kinase, and ultimately decreased glucose transport [1,9]. Selective activation of muscle PKCh has been associated with dietary-induced models of insulin resistance [2,8] and by lipid infusion in rats leading to a downregulation of insulin signaling [3]. In addition, PKCh has been shown to disrupt insulin-induced IRS-1 tyrosine phosphorylation 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.02.177 * Corresponding author. E-mail address: cristina.rondinone@roche.com (C.M. Rondinone). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 343 (2006) 361–368 BBRC