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

Abstract

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.

Defining the role of DAG, mitochondrial function, and lipid deposition in palmitate-induced proinflammatory signaling and its counter-modulation by palmitoleate

“…Toll-like receptors (TLR) of the innate immune system, in particular TLR2 and TLR4 (for which SFAs may be ligands), have been implicated in the activation of NF B ( 20,21 ), but there is also considerable evidence showing that members of the protein kinase C (PKC) ( 22,23 ) and MAP kinase families ( 7,18 ) can promote SFA-induced NF B signaling independently of TLR involvement in skeletal muscle cells. We recently reported that palmitate induces activation of JNK, p38 MAPK, and MEK-ERK signaling but that only pharmacological inhibition of the MEK-ERK pathway attenuated SFA-induced NF B signaling and IL-6 expression in L6 skeletal muscle cells ( 18 ).…”

Proteomic Analysis of the Palmitate-induced Myotube Secretome Reveals Involvement of the Annexin A1-Formyl Peptide Receptor 2 (FPR2) Pathway in Insulin Resistance*

“…Elevated free fatty acid levels in the plasma of obese patients play an important role in the development of insulin resistance (6). Hence, lowering the free fatty acid level in plasma has been shown to restore insulin sensitivity in these patients (7). Palmitate (C16:0) is a saturated free fatty acid found in animal plasma.…”

Palmitate Contributes to Insulin Resistance through Downregulation of the Src-Mediated Phosphorylation of Akt in C2C12 Myotubes

“…Experimental plasma FFA elevation impairs glucose oxidation/glycogen synthesis and decreases glucose transport, which leads to organ-specific insulin resistance in adipocytes, skeletal muscle cells, and hepatocytes, 5,6) whereas lowering FFA levels in vivo and vitro significantly improves insulin sensitivity. 7,8) Prolonged exposure to elevated FFA results in the production of inflammatory factors by stimulating the nuclear factor-B (NF-B) pathway, [9][10][11] and induces activation of Jun N-terminal kinase-3 (JNK3), which accelerates -oxidation of FFA, brings about excessive electron flux in the mitochondrial respiratory chain, and subsequently causes increased reactive oxidant species (ROS) generation. 5,12,13) All of this has passive effects on the insulin signal transduction pathway, leading to decreased activity of phosphatidylinositol-3 kinase (PI3K), reduced phosphorylation of the three known isoforms of Akt, and impaired translocation of glucose transporter-4 (Glut4), finally resulting in inhibited glucose uptake and utilization.…”