siRNA-mediated reduction of inhibitor of nuclear factor-kappaB kinase prevents tumor necrosis factor-alpha-induced insulin resistance in human skeletal muscle.

Abstract

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-kappaB kinase (IKK)beta in TNF-alpha-induced impairments in insulin signaling and glucose metabolism in skeletal muscle. Small interfering RNA (siRNA) was used to silence IKKbeta gene expression in primary human skeletal muscle myotubes from nondiabetic subjects. siRNA gene silencing reduced IKKbeta protein expression 73% (P < 0.05). Myotubes were incubated in the absence or presence of insulin and/or TNF-alpha, and effects of IKKbeta silencing on insulin signaling and glucose metabolism were determined. 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. IKKbeta 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 IKKbeta reduction. siRNA-mediated IKKbeta reduction prevented TNF-alpha-induced insulin resistance on Akt Ser(473) and Thr(308) phosphorylation and phosphorylation of the 160-kDa Akt substrate AS160. IKKbeta 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) was unaltered by IKKbeta siRNA. IKKbeta silencing prevents TNF-alpha-induced impairments in insulin action on Akt phosphorylation and glucose uptake and metabolism in human skeletal muscle.