Lipid induced overexpression of NF-κB in skeletal muscle cells is linked to insulin resistance

Abstract

Lipid induced NF-κB activation is known to be associated with insulin resistance and type2 diabetes. Here we show that incubation of L6 skeletal muscle cells with palmitate significantly increased NF-κB p65 and NF-κB p50 expression along with their phosphorylation. NF-κB p65 siRNA inhibited palmitate induced overexpression of NF-κB p65 indicating palmitate effect on transcriptional activation. RT-PCR and real time PCR experiments also showed a significant increase in NF-κB p65 gene expression due to palmitate. Overexpression of NF-κB p65 by palmitate was linked to impairment of insulin activity. Palmitate effect on NF-κB gene and protein expression was found to be mediated by phospho-PKC^ε as calphostin C (an inhibitor of PKC) and ^εV1 (PKC^ε translocation inhibitor) significantly reduced NF-κB expression. To understand the underlying mechanism, we purified NF-κB and pPKC^ε from palmitate incubated skeletal muscle cells and their interaction in cell free system demonstrated the transfer of phosphate from PKC^ε to NF-κB. This prompted us to transduct pPKC^ε to the skeletal muscle cells. These cells showed increased amount of pNF-κB and NF-κB. Excess of NF-κB p65 pool thus created in the cells made them insulin resistant. Addition of NF-κB p65 siRNA and SN50 inhibited palmitate induced NF-κB p65 expression indicating NF-κB regulation of its gene expression. Increase of NF-κB did not affect the activation of IKK/IκB indicating NF-κB p65 expression to be a distinct effect of palmitate. Since NF-κB p65 is linked to several diseases, including type2 diabetes, this report may be important in understanding the pathogenicity of these diseases.