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

Abstract

OBJECTIVE-Proinflammatory cytokines contribute to systemic low-grade inflammation and insulin resistance. Tumor necrosis factor (TNF)-␣ impedes insulin signaling in insulin target tissues. We determined the role of inhibitor of nuclear factor-B kinase (IKK)␤ 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 IKK␤ gene expression in primary human skeletal muscle myotubes from nondiabetic subjects. siRNA gene silencing reduced IKK␤ protein expression 73% (P Ͻ 0.05). Myotubes were incubated in the absence or presence of insulin and/or TNF-␣, and effects of IKK␤ silencing on insulin signaling and glucose metabolism were determined.RESULTS-Insulin increased glucose uptake 1.7-fold (P Ͻ 0.05) and glucose incorporation into glycogen 3.8-fold (P Ͻ 0.05) in myotubes from nondiabetic subjects. TNF-␣ exposure fully impaired insulin-mediated glucose uptake and metabolism. IKK␤ siRNA protected against TNF-␣-induced impairments in glucose metabolism, since insulin-induced increases in glucose uptake (1.5-fold; P Ͻ 0.05) and glycogen synthesis (3.5-fold; P Ͻ 0.05) were restored. Conversely, TNF-␣-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␤ reduction. siRNA-mediated IKK␤ reduction prevented TNF-␣-induced insulin resistance on Akt Ser 473 and Thr 308 phosphorylation and phosphorylation of the 160-kDa Akt substrate AS160. IKK␤ 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 IKK␤ siRNA.CONCLUSIONS-IKK␤ silencing prevents TNF-␣-induced impairments in insulin action on Akt phosphorylation and glucose uptake and metabolism in human skeletal muscle.

Specificity of insulin signalling in human skeletal muscle as revealed by small interfering RNA

“…These signal transducers act as inhibitory molecules, which provide negative feedback on canonical insulin signalling and impair glucose uptake and metabolism volunteers to TNF-α. We next used siRNA directed against IKKβ (also known as IKBKB) [52] or MAP4K4 [53]-the protein products of which were identified by Czech and coworkers [41] as regulators of cellular GLUT4 protein content and/or membrane trafficking in cultured 3T3-L1 adipocytes-to investigate whether targeted depletion of these proteins would prevent the development of insulin resistance. Recent results from our laboratory related to the role of IKKβ [52] and MAP4K4 [53] will be reviewed below.…”

“…We next used siRNA directed against IKKβ (also known as IKBKB) [52] or MAP4K4 [53]-the protein products of which were identified by Czech and coworkers [41] as regulators of cellular GLUT4 protein content and/or membrane trafficking in cultured 3T3-L1 adipocytes-to investigate whether targeted depletion of these proteins would prevent the development of insulin resistance. Recent results from our laboratory related to the role of IKKβ [52] and MAP4K4 [53] will be reviewed below. Since defects in skeletal muscle glucose transport and cell surface GLUT4 content in type 2 diabetic patients correlate with impaired whole body insulin-mediated glucose uptake [54][55][56], targeting these novel signalling cascades that regulate GLUT4 or translocation may be efficacious in the treatment of whole body insulin resistance.…”

“…Using primary human skeletal muscle myotubes we determined the effect of IKKβ gene silencing by siRNA on insulin action [52]. Myotubes were then incubated in the absence or presence of TNF-α for 2 h. Exposure to TNF-α impaired the action of insulin on glucose uptake and metabolism and reduced the phosphorylation of AKT and the AKT substrate AS160 [52].…”

“…Myotubes were then incubated in the absence or presence of TNF-α for 2 h. Exposure to TNF-α impaired the action of insulin on glucose uptake and metabolism and reduced the phosphorylation of AKT and the AKT substrate AS160 [52]. Gene silencing reduced IKKβ protein levels by 75%, without affecting cell differentiation or the expression of GLUT1 or GLUT4 (also known as SLC2A1 and SLC2A4, respectively) mRNAs.…”

Of rodents and men: Species-specific glucose regulation and type 2 diabetes research

“…events such as paracrine signaling can also be detected in real time using biosensor cells in the vicinity of isolated human islets (Rodriguez-Diaz et al, 2012). Minimally invasive techniques involving phlebotomy and muscle biopsies can be utilized for various biochemical and systemic regulation analyses (Chavez et al, 2009;Zierath et al, 2000), and gene-silencing studies can be extended to human skeletal muscle samples (Austin et al, 2008;Bouzakri and Zierath, 2007). the use of human organs ex vivo facilitates the integration of cellular data to the organ level.…”