Egg White Ovotransferrin‐Derived ACE Inhibitory Peptide Ameliorates Angiotensin II‐Stimulated Insulin Resistance in Skeletal Muscle Cells

Figure 1. Effects of egg white ovotransferrin‐derived tripeptides on cell viability and glucose uptake in L6 myotubes. A) Effects of IRW, IQW, or LKP on cell viability. Fully differentiated myotubes were incubated with 100 μm of IRW, IQW, or LKP 2 h prior to the treatment of 1 μm of Ang II for 24 h. Cell viability was examined using MTT assay. B) Effects of IRW, IQW, or LKP on insulin‐independent glucose uptake. Fully differentiated L6 myotubes were preincubated in 24‐place multiwell plates in Krebs‐Henseleit‐Hepes buffer (KHH buffer) without glucose for 2 h. They were then incubated in KHH buffer containing 11 mm glucose without or with insulin and 100 μm of peptides for 4 h. C) Effect of peptides on insulin stimulated glucose uptake in Ang II‐treated L6 myotubes. Fully differentiated myotubes were incubated with 100 μm of IRW, IQW, or LKP 2 h prior to the treatment of 1 μm of Ang II for 24 h. Next, the myotubes were kept for 2 h in KHH buffer. The myotubes were then cultured in KHH buffer containing 11 mm glucose in the absence or presence of 100 nm of insulin for another 4 h, and then the glucose uptake was measured using a Glucose CII Test Kit. Each value represents the mean ± SEM of…

Figure 2. Effects of egg white ovotransferrin‐derived tripeptides on insulin signaling pathway in Ang II treated L6 myotubes. Fully differentiated myotubes were treated with 100 μm of peptides for 2 h followed by treatment with 1 μm of Ang 2 for 24 h. L6 myotubes were preincubated in KHH buffer for 2 h. They were then incubated in KHH buffer containing 11 mm glucose without or with 100 nm of insulin for 30 min. The cells were lysed and western blotting of the lysates was performed with antibodies against p‐IRS‐1 Ser 307 (A), p‐IRS‐1 tyr 632 (B), total IRS‐1 (A,B), p‐Akt (C), total Akt (C), and α‐tubulin (loading control). A set of representative images is shown. Data are presented as mean ± SEM of three independent experiments. **indicates p < 0.05 as compared to the Ang II‐treated group. *indicates p < 0.05 as compared to the untreated group.

Figure 3. Effects of egg white ovotransferrin‐derived tripeptides (peptides) on GLUT4 translocation in L6 myotubes. Fully differentiated myotubes were treated with 100 μm of peptides for 2 h followed by treatment with 1 μm of Ang II for 24 h. L6 myotubes were preincubated in KHH buffer for 2 h. They were then incubated in KHH buffer containing 11 mm glucose without or with 100 nm of Insulin for 30 min. A) GLUT4 localization was detected using immunofluorescence technique and western blotting. Cellular localization of GLUT4 proteins is shown in red fluorescence (left) and merged image is also shown (right). A representative set of images from three independent experiments is shown. B) The membrane and cytosol were separated from the cells. The expression of GLUT4 was tested by western blot. Each value represents the mean ± SEM of three independent experiments. **indicates p < 0.05 as compared to the Ang II‐treated group. *indicates p < 0.05 as compared to the untreated group.

Figure 4. Effects of egg white ovotransferrin‐derived tripeptides on AT1R and AT2R expression in Ang II treated L6 myotubes. Fully differentiated myotubes were treated with 100 μm of peptides for 2 h followed by the treatment with 1 μm of Ang II for 24 h. The cells were lysed and western blotting of the lysates was performed with antibodies against AT1R A–D), AT2R E), and α‐tubulin (loading control). A set of representative images is shown. Data are presented as mean ± SEM of three independent experiments. **indicates p < 0.05 as compared to the Ang II‐treated group.

Figure 5. Effects of egg white ovotransferrin derived tripeptides on Ang II induced ROS in L6 myotubes. L6 myotubes were pretreated with 100 μm of Peptides for 2 h prior to 30 min stimulation with 1 μm of Ang II. Cells were treated with 10 μm of DHE for 30 min, and then visualized by fluorescent microscopy. The mean fluorescence intensity was calculated, and the data were expressed as % of the Ang II‐treat group from four independent experiments. Mean ± SEM are shown. **indicates p < 0.05 as compared to the Ang II‐treated group. A representative set of images from three independent experiments is shown.

Figure 6. Effects of egg white ovotransferrin‐derived tripeptides on p67phox and p47phox translocation in L6 myotubes. L6 myotubes were pretreated with 100 μm of Peptides for 2 h followed by 30 min stimulation with 1 μm of Ang II. The membrane and cytosol were separated from the cells. The expression of p67phox (A) and p47phox (B) were tested by western blotting. The data are expressed as % of the Ang II‐treated group from four independent experiments. Mean ± SEM are shown. **indicates p < 0.05 as compared to the Ang II‐treated group.