siRNA-Mediated Reduction of Inhibitor of Nuclear Factor-κB Kinase Prevents Tumor Necrosis Factor-α–Induced Insulin Resistance in Human Skeletal Muscle

siRNA-Mediated Reduction of Inhibitor of Nuclear Factor-κB Kinase Prevents Tumor Necrosis Factor-α–Induced Insulin Resistance in Human Skeletal Muscle Reginald L. Austin 1 , Anna Rune 1 , Karim Bouzakri 1 , Juleen R. Zierath 1 and Anna Krook 1 2 1 Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden 2 Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden Corresponding author: Anna Krook, anna.krook{at}ki.se Abstract OBJECTIVE— Proinflammatory cytokines contribute to systemic low-grade inflammation and insulin resistance. Tumor necrosis factor (TNF)-α impedes insulin signaling in insulin target tissues. We determined the role of inhibitor of nuclear factor-κB kinase (IKK)β in TNF-α–induced impairments in insulin signaling and glucose metabolism in skeletal muscle. RESEARCH DESIGN AND METHODS— Small interfering RNA (siRNA) was used to silence IKKβ gene expression in primary human skeletal muscle myotubes from nondiabetic subjects. siRNA gene silencing reduced IKKβ protein expression 73% ( P < 0.05). Myotubes were incubated in the absence or presence of insulin and/or TNF-α, and effects of IKKβ 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-α exposure fully impaired insulin-mediated glucose uptake and metabolism. IKKβ siRNA protected against TNF-α–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-α–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 IKKβ reduction. siRNA-mediated IKKβ reduction prevented TNF-α–induced insulin resistance on Akt Ser 473 and Thr 308 phosphorylation and phosphorylation of the 160-kDa Akt substrate AS160. IKKβ 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 IKKβ siRNA. CONCLUSIONS— IKKβ silencing prevents TNF-α–induced impairments in insulin action on Akt phosphorylation and glucose uptake and metabolism in hu