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

Abstract

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-kappaB 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 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.

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. 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.

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

FIG. 1.

Protein expression of IKKβ and IκBα. A: Protein expression of IKKβ was unaltered in cells exposed to a scrambled siRNA sequence, a 2-h incubation with TNF-α (lanes 1–3). siRNA against IKKβ reduced protein expression by 73% (lanes 4 and 5). Glyceraldehyde-3-phosphate dehydrogenase (GADPH) expression was determined to control for equal loading. B: IκBα was measured after siRNA-mediated depletion of IKKβ. Results are means ± SE. *P < 0.05 vs. transfected scrambled siRNA (Scr) control (n = 4).

FIG. 2.

Myotube growth. A: Morphological appearance of human skeletal muscle myotube formation after introduction of scrambled siRNA or siRNA against IKKβ. Photomicrographs are shown at 10× magnification for myotubes at day 6 of differentiation (4 days after introduction of siRNA). Results are from two separate experiments. B: Representative immunoblot showing the differentiation marker desmin.

FIG. 3.

Effect of siRNA-mediated silencing of IKKβ on glucose uptake and glycogen synthesis. Glucose uptake (A) and glycogen synthesis (B) were measured in differentiated primary skeletal muscle myotubes under basal conditions (□) and after insulin stimulation (▪). Results are means ± SE. *P < 0.05 vs. respective basal cells. †P < 0.05 vs. TNF-α–and insulin-stimulated untransfected cells (n = 4).

FIG. 4.

Akt Ser⁴⁷³ and Thr³⁰⁸ phosphorylation. Representative immunoblot showing Akt Ser⁴⁷³ (A) and Akt Thr³⁰⁸ (B) phosphorylation in response to insulin and TNF-α. C: Total Akt protein expression. Graphs are summarized data (mean ± SE). *P < 0.05% vs. untransfected control cells. †P < 0.05 vs. TNF-α–stimulated untransfected cells (n = 9).

FIG. 5.

GSK-3β and AS160 phosphorylation. Representative immunoblot showing pGSK3β Ser⁹ (A) and Akt substrate AS160 (B) phosphorylation in response to insulin or TNF-α under basal conditions (□) and after insulin stimulation (▪). Graphs are summarized data (mean ± SE). *P < 0.05 vs. respective basal. †P < 0.05 vs. TNF-α–stimulated untransfected cells. #P < 0.05 vs. control basal (GSK3β, n = 3; AS160, n = 4).

FIG. 6.

Effect of siRNA-mediated silencing of IKKβ on ERK1/2 MAPK phosphorylation. A: Representative immunoblot of ERK1/2 MAPK phosphorylation. B: Total ERK-2 protein expression. C: Summarized results for ERK1/2 MAPK under basal (□) or insulin-stimulated (▪) conditions. (n = 5). D: Glycogen synthesis was measured in differentiated primary skeletal muscle myotubes following 1 h of exposure to 50 μmol/l PD98059. Results are means ± SE from experiment carried out in triplicate. *P < 0.05 vs. respective basal. #P < 0.05 vs. control basal.

FIG. 7.

Effect of siRNA-mediated silencing of IKKβ on JNK phosphorylation. A: Representative immunoblot showing p54/p46 JNK phosphorylation. B: Summarized results for p54 and p46 JNK phosphorylation under basal (□) or insulin-stimulated (▪) conditions. Results are mean ± SE. *P < 0.05 vs. respective basal. #P < 0.05 vs. control basal (n = 3).

FIG. 8.

Effect of siRNA-mediated silencing of IKKβ on IRS-1 Ser³¹² phosphorylation. Representative immunoblot showing IRS-1 migration on SDS-PAGE (A) or IRS-1 Ser³¹² phosphorylation (B). IRS-1 Ser³¹² phosphorylation results under basal (□) and insulin-stimulated (▪) conditions are reported as means ± SE percent relative to basal untransfected controls. Arrow highlights mobility shift (A) (n = 4).