AVE0991, a Nonpeptide Angiotensin 1-7 Receptor Agonist, Improves Glucose Metabolism in the Skeletal Muscle of Obese Zucker Rats: Possible Involvement of Prooxidant/Antioxidant Mechanisms

Figures

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  Figure 1. Intraperitoneal glucose tolerance test (IPGTT) evaluated in control obese Zucker rats (n = 6) and in obese Zucker rats receiving AVE0991 (n = 5) via osmotic minipumps for two weeks. AUC: area under curve. Data are presented as mean ± S.E.M. and were analysed using Student's t-test, ∗p < 0.05; ∗∗p < 0.01.

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  Figure 2. Classical RAS pathway components in the skeletal muscle of obese Zucker rats. Gene expression of renin, angiotensinogen (AGT), angiotensin-converting enzyme (ACE), angiotensin II type 1 receptor (AT1), and angiotensin II type 2 receptor (AT2) in musculus quadriceps of control obese Zucker rats and in obese Zucker rats receiving AVE0991 via osmotic minipumps for two weeks, determined by real-time PCR. Data were normalized to the gene expression of 40S ribosomal protein S29 (Rps29) whose expression was not altered by the treatment. Data presented as mean ± S.E.M. were analysed by Student's t-test.

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  Figure 3. Alternative RAS pathway components in the skeletal muscle of obese Zucker rats. Gene expression of angiotensin-converting enzyme2 (ACE2), Mas receptor, and neutral endopeptidase (NEP) in musculus quadriceps of control obese Zucker rats and in obese Zucker rats receiving AVE0991 via osmotic minipumps for two weeks, determined by real-time PCR. Data were normalized to the gene expression of 40S ribosomal protein S29 (Rps29) whose expression was not altered by the treatment. Data presented as mean ± S.E.M. were analysed by Student's t-test.

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  Figure 4. Plasma and skeletal muscle APA activity. Enzyme activity of aminopeptidase A (APA) measured in the membrane fraction isolated from the skeletal muscle and the blood plasma control obese Zucker rats and in obese Zucker rats receiving AVE0991 via osmotic minipumps for two weeks. Gene expression of aminopeptidase A (APA) in musculus quadriceps determined by real-time PCR. Data were normalized to the gene expression of 40S ribosomal protein S29 (Rps29) whose expression was not altered by the treatment. Data presented as mean ± S.E.M. were analysed by Student's t-test, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

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  Figure 5. Renin receptor in the skeletal muscle of Zucker rats. Gene expression of the renin receptor (ReR) at the level of mRNA (a) and protein with representative Western blots (b); mRNA expression of the transcription factor promyelocytic leukemia zinc finger (PLZF, Zbtb16) and furin in musculus quadriceps of control obese Zucker rats and in obese Zucker rats receiving AVE0991 via osmotic minipumps for two weeks. Levels of mRNA were determined by real-time PCR. Data were normalized to the gene expression of 40S ribosomal protein S29 (Rps29) whose expression was not altered by the treatment. The amount of renin receptor protein was quantified by Western blot method. The signal intensities of the proteins of interest were normalized to the sample's total protein content stained with Coomassie Brilliant Blue. Data are presented as mean ± S.E.M. Results were analysed by Student's t-test, ∗p < 0.05.

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  Figure 6. Prooxidant genes in the skeletal muscle of obese Zucker rats. Gene expression of NADPH oxidase 2 (Nox2), Nox4, and p22phox in musculus quadriceps of control obese Zucker rats and in obese Zucker rats receiving AVE0991 via osmotic minipumps for two weeks, determined by real-time PCR. Data were normalized to the gene expression of 40S ribosomal protein S29 (Rps29) whose expression was not altered by the treatment. Data presented as mean ± S.E.M. were analysed by Student's t-test, ∗p < 0.05; ∗∗p < 0.01.

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  Figure 7. Genes coding enzymes with antioxidant action in the skeletal muscle of obese Zucker rats. Gene expression of superoxide dismutase 1 (Sod1), Sod2, Sod3, and nuclear factor erythroid 2-related factor 2 (Nrf2) in musculus quadriceps of control obese Zucker rats and in obese Zucker rats receiving AVE0991 via osmotic minipumps for two weeks, determined by real-time PCR. Data were normalized to the gene expression of 40S ribosomal protein S29 (Rps29) whose expression was not altered by the treatment. Data presented as mean ± S.E.M. were analysed by Student's t-test, ∗p < 0.05; ∗∗p < 0.01.

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  Figure 8. Insulin signaling cascade in the skeletal muscle of obese Zucker rats. Protein content of total insulin receptor β subunit (IRβ), phosphorylated IRβ at Tyr1150/1151, total insulin receptor substrate 1 (IRS1), and phosphorylated IRS1 at Tyr896, Ser307, and Ser612 with representative Western blots in musculus quadriceps of control obese Zucker rats and in obese Zucker rats receiving AVE0991 via osmotic minipumps for two weeks. The amount of protein was quantified by Western blot method. The signal intensities of the proteins of interest were normalized to the sample's total protein content stained with Coomassie Brilliant Blue. Data are presented as mean ± S.E.M. Results were analysed by Student's t-test, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

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  Figure 9. Protein tyrosine phosphatase 1B (PTP1B) in the skeletal muscle of obese Zucker rats. Protein content of total protein tyrosine phosphatase 1B (PTP1B), phosphorylated PTP1B at Ser50 with representative Western blots, and PTP1B activity in musculus quadriceps of control obese Zucker rats and in obese Zucker rats receiving AVE0991 via osmotic minipumps for two weeks. The amount of protein was quantified by Western blot method, and PTP1B activity was measured using commercially available colorimetric assay kit. The signal intensities of the proteins of interest were normalized to the sample's total protein content stained with Coomassie Brilliant Blue. Data are presented as mean ± S.E.M. Results were analysed by Student's t-test, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.