SiRNA-mediated reduction of inhibitor of nuclear factor-κB kinase prevents tumor necrosis factor-α-induced insulin resistance in human skeletal muscle

OBJECTIVE-Proinflammatory cytokines contribute to systemic low-grade inflammation and insulin resistance. Tumor necrosis factor (TNF)-alpha impedes insulin signaling in insulin target tissues. We determined the role of inhibitor of nuclear factor-kappa B kinase (IKK)beta in TNF-alpha-induced impairments in insulin signaling and glucose metabolism in skeletal muscle. RESEARCH DESIGN AND METHODS-Small interfering RNA (siRNA) was used to silence IKK beta gene expression in primary human skeletal muscle myotubes from nondiabetic subjects. siRNA gene silencing reduced IKK beta protein expression 73% (P < 0.05). Myotubes were incubated in the absence or presence of insulin and/or TNF-alpha, and effects of IKK beta silencing on insulin signaling and glucose metabolism were determined. RESULTS-Insulin increased glucose uptake 1.7-fold (P < 0.05) and glucose incorporation into glycogen 3.8-fold (P < 0.05) in myotubes from nondiabetic subjects. TNF-alpha exposure fully impaired insulin-mediated glucose uptake and metabolism. IKK beta siRNA protected against TNF-alpha-induced impairments in glucose metabolism, since insulin-induced increases in glucose uptake (1.5-fold; P < 0.05) and glycogen synthesis (3.5-fold; P < 0.05) were restored. Conversely, TNF-alpha-induced increases in insulin receptor substrate-1 serine phosphorylation (Ser(312)), Jun NH(2)-terminal kinase phosphorylation, and extracellular signal-related kinase-1/2 mitogen-activated protein kinase (MAPK) phosphorylation were unaltered by siRNA-mediated IKK beta reduction. siRNA-mediated IKK beta reduction prevented TNF-alpha-induced insulin resistance on Akt Ser(473) and Thr(308) phosphorylation and phosphorylation of the 160-kDa Akt substrate AS160. IKK beta silencing had no effect on cell differentiation. Finally, mRNA expression of GLUT1 or GLUT4 and protein expression of MAPK kinase kinase kinase isoform 4 (MAP4K4(4) was unaltered by IKK beta siRNA. CONCLUSIONS-IKK beta silencing prevents TNF-alpha-induced impairments in insulin action on Akt phosphorylation and glucose uptake and metabolism in human skeletal muscle.