Volume 4, Issue 1, Pages 89-96 (July 2006) siRNA-based gene silencing reveals specialized roles of IRS-1/Akt2 and IRS-2/Akt1 in glucose and lipid metabolism in human skeletal muscle  Karim Bouzakri, Anna Zachrisson, Lubna Al-Khalili, Bei B. Zhang, Heikki A. Koistinen, Anna Krook, Juleen R. Zierath  Cell Metabolism  Volume 4, Issue 1, Pages 89-96 (July 2006) DOI: 10.1016/j.cmet.2006.04.008 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Silencing of IRS-1, IRS-2, Akt1, and Akt2 in myoblasts or myotubes A) Morphological changes of human skeletal muscle after silencing of IRS-1 and Akt2 in myoblastes. B) Representative immunoblot showing protein expression of IRS-1, IRS-2 Akt1, Akt2, or desmin after silencing of the respective targets in myotubes. Graph shows mean ± SEM presented as arbitrary units relative to control cells incubated under basal conditions. ∗p < 0.05 versus untranfected cells. Cell Metabolism 2006 4, 89-96DOI: (10.1016/j.cmet.2006.04.008) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Insulin action on signal transduction Representative immunoblot highlighting the role of IRS-1, IRS-2, Akt1, or Akt2 on phosphorylation of (A) Akt Ser473, (B) Akt Thr308 (C) AS160, (D) FoxO1/FoxO4, and (E) ERK1/2 MAPK under basal (open box) or insulin (closed box) treatment. Graph shows mean ± SEM arbitrary units relative to control cells under basal conditions. ∗p < 0.05 versus untransfected cells at basal, #p < 0.05 versus untransfected cells after insulin stimulation. Cell Metabolism 2006 4, 89-96DOI: (10.1016/j.cmet.2006.04.008) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Glucose uptake, glycogen synthesis, palmitate uptake, and oxidation Glucose uptake (A) was measured on differentiated primary human skeletal muscle myotubes under basal conditions (open box) and after insulin stimulation (closed box). Measurements were performed after silencing of IRS-1, IRS-2, Akt1, or Akt2. Glycogen synthesis (B) was assessed under basal (open box) and insulin (closed box) treatment after silencing of the target genes. Glucose uptake and glycogen synthesis are reported as cpm/mg protein/min ± SEM. Effect of IRS-1, IRS-2, Akt1, and Akt2 on palmitate (C) uptake and (D) oxidation under basal (open box) or insulin (closed box) treatment in differentiated primary human skeletal muscle myotubes. Palmitate uptake and oxidation are reported as pmol/mg protein/min. Data is mean ± SEM. ∗p < 0.05 versus untransfected cells at basal, #p < 0.05 versus untransfected cells after insulin stimulation. Cell Metabolism 2006 4, 89-96DOI: (10.1016/j.cmet.2006.04.008) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Schematic view of signaling pathways regulating myotube formation and glucose/lipid metabolism Cell Metabolism 2006 4, 89-96DOI: (10.1016/j.cmet.2006.04.008) Copyright © 2006 Elsevier Inc. Terms and Conditions