Lipid induced overexpression of NF-kappaB in skeletal muscle cells is linked to insulin resistance.
Lipid induced NF-kappaB 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-kappaB p65 and NF-kappaB p50 expression along with their phosphorylation. NF-kappaB p65 siRNA inhibited palmitate induced overexpression of NF-kappaB p65 indicating palmitate effect on transcriptional activation. RT-PCR and real time PCR experiments also showed a significant increase in NF-kappaB p65 gene expression due to palmitate. Overexpression of NF-kappaB p65 by palmitate was linked to impairment of insulin activity. Palmitate effect on NF-kappaB gene and protein expression was found to be mediated by phospho-PKCepsilon as calphostin C (an inhibitor of PKC) and epsilonV1 (PKCepsilon translocation inhibitor) significantly reduced NF-kappaB expression. To understand the underlying mechanism, we purified NF-kappaB and pPKCepsilon from palmitate incubated skeletal muscle cells and their interaction in cell free system demonstrated the transfer of phosphate from PKCepsilon to NF-kappaB. This prompted us to transduct pPKCepsilon to the skeletal muscle cells. These cells showed increased amount of pNF-kappaB and NF-kappaB. Excess of NF-kappaB p65 pool thus created in the cells made them insulin resistant. Addition of NF-kappaB p65 siRNA and SN50 inhibited palmitate induced NF-kappaB p65 expression indicating NF-kappaB regulation of its gene expression. Increase of NF-kappaB did not affect the activation of IKK/IkappaB indicating NF-kappaB p65 expression to be a distinct effect of palmitate. Since NF-kappaB p65 is linked to several diseases, including type2 diabetes, this report may be important in understanding the pathogenicity of these diseases.
Barma
Pomy
P
Molecular Endocrinology Laboratory, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata - 700032, India.
Bhattacharya
Sushmita
S
Bhattacharya
Anirban
A
Kundu
Rakesh
R
Dasgupta
Suman
S
Biswas
Anindita
A
Bhattacharya
Shelley
S
Roy
Sib Sankar
SS
Bhattacharya
Samir
S
D000818:Animals; D002460:Cell Line; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism; D007333:Insulin Resistance / Q000502:physiology*; D050356:Lipid Metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D052002:NF-kappa B p50 Subunit / Q000235:genetics / Q000378:metabolism*; D010168:Palmitates / Q000378:metabolism*; D010766:Phosphorylation; D051744:Protein Kinase C-epsilon / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*
D052002:NF-kappa B p50 Subunit; D010168:Palmitates; D034741:RNA, Small Interfering; D051996:Transcription Factor RelA; D051744:Protein Kinase C-epsilon