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siRNA-Mediated Reduction of IKKβ Prevents TNF--Induced Insulin Resistance in Human Skeletal Muscle

¹Department of Molecular Medicine and Surgery, and Department of Physiology and
²Pharmacology, Karolinska Institutet S-171 77 Stockholm

Objective: Pro-inflammatory cytokines contribute to systemic low-grade inflammation and insulin resistance. Tumor necrosis factor-alpha (TNF-) impedes insulin signaling in insulin target tissues. We determined the role of IKKβ in TNF--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 non-diabetic 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- and effects of IKKβ silencing were determined on insulin signaling and glucose metabolism.

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 non-diabetic subjects. TNF- exposure fully impaired insulin-mediated glucose uptake and metabolism. IKKβ siRNA protected against TNF--induced impairments in glucose metabolism, since insulin-induced increases in glucose uptake uptake (1.5-fold, p<0.05) and glycogen synthesis (3.5-fold, p<0.05) were restored. Conversely, TNF--induced increases in insulin receptor substrate-1 (IRS-1) serine phosphorylation (Ser³¹²), JNK or ERK-1/2 MAPK phosphorylation were unaltered by siRNA-mediated IKKβ reduction. siRNA-mediated IKKβ reduction also prevented TNF--induced insulin resistance on Akt Ser⁴⁷³ and Thr³⁰⁸ phosphorylation and phosphorylation of the 160kDa Akt substrate AS160. IKKβ silencing was without effect on cell differentiation. Finally, mRNA expression of GLUT1, GLUT4, or protein expression of mitogenic-activated protein kinase kinase kinase kinase isoform 4 (MAP4K4) was unaltered by IKKβ siRNA.

Conclusion: IKKβ silencing prevents TNF--induced impairments in insulin action on Akt phosphorylation and glucose uptake and metabolism in human skeletal muscle.

Key Words: Human Skeletal Muscle • Cell Culture • Insulin Resistance • Tumor Necrosis Factor Alpha • IB Kinase Beta

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