ACE ‐inhibitory peptide Leu‐Tyr‐Ala in skeletal muscle actin exhibits anti‐aging effects through the inhibition of acn‐1 in Caenorhabditis elegans

Figures

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  Figure 1. ACE‐inhibitory and antioxidant activities of peptides derived from skeletal muscle actin. (A) ACE‐inhibitory activity of each peptide (5 mmol/L) and captopril (0.1 mmol/L). (B) Antioxidant activity of Leu‐Tyr‐Ala at 5 mmol/L. Data are expressed as mean ± SEM (n = 3). Ascorbic acid was used as the positive control for antioxidant activity. Different letters indicate significant differences in ACE‐inhibitory activity (P < 0.05, Tukey–Kramer test).

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  Figure 2. ACE‐inhibitory activity of four peptides at different concentrations for the calculation of IC50 values. Mean ± SEM (n = 3).

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  Figure 3. Effects of ACE inhibitors on body length and width of Caenorhabditis elegans. (A) Changes in body length of wild‐type (N2) C. elegans and acn‐1 (tm4241) mutants treated with ACE inhibitors. (B) Changes in body width of wild‐type (N2) C. elegans and acn‐1 (tm4241) mutants treated with ACE inhibitors. (C) Representative images of nematodes treated with ACE inhibitors on day 4. (D) Body length on day 10 from adulthood. Data are expressed as mean ± SEM ((A, B), n = 30; (D), n = 12). Statistical analysis was performed using Tukey–Kramer test. Different letters indicate significant differences (P < 0.05).

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  Figure 4. Effects of Leu‐Tyr‐Ala on reproductive capacity of Caenorhabditis elegans. (A) Changes in progeny number of wild‐type (N2) C. elegans treated with Leu‐Tyr‐Ala. (B) Total progeny number per nematode. Data are expressed as mean ± SEM (n = 10). Statistical analysis was performed using two‐tailed t‐test.

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  Figure 5. Effects of Leu‐Tyr‐Ala on lifespan, locomotor activity, stress resistance, and lipofuscin. (A) Changes in lifespan of wild‐type (N2) Caenorhabditis elegans treated with captopril (2.5 mmol/L), or Leu‐Tyr‐Ala (0.5 and 5 mmol/L). (B) Locomotor activity. (C) Survival rate of nematodes exposed to 5 mmol/L H2O2 (D) Survival rate of nematodes exposed to heat stress (37 °C) after 10 h. (E) Representative images of lipofuscin autofluorescence intensity. (F) Quantification of lipofuscin. Data are expressed as mean ± SEM (lifespan assay and stress resistance assays, n = 3; locomotor activity assay, n = 15 per day; lipofuscin assay, n = 15). Distilled water was used as the control. For statistical analysis, the log‐rank test was used for lifespan and oxidative stress assays (*P < 0.05, **P < 0.01); Tukey–Kramer test was applied for locomotor activity, thermal stress, and lipofuscin assays. Different letters indicate significant differences (P < 0.05).

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  Figure 6. Effects of Leu‐Tyr‐Ala on fat accumulation in wild‐type Caenorhabditis elegans. (A) Representative images of nematodes stained by Oil Red O (ORO). (B) Quantification of ORO intensity. Data are expressed as mean ± SEM (n = 30). Tukey–Kramer test was used to determine significant differences (*P < 0.05). Values of P < 0.1 were considered marginally significant. Distilled water was used as the control.

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  Figure 7. Changes in the mRNA expression levels after Leu‐Tyr‐Ala treatment in wild‐type Caenorhabditis elegans. (A) Change in acn‐1 mRNA expression level; (B) changes in mRNA expression levels of muscle related genes; (C) mRNA levels of daf‐16‐related genes; (D) mRNA levels of daf‐12‐related genes. Data are expressed as mean ± SEM (n = 4). Two‐tailed Student's t‐test was performed to determine significant differences (*P < 0.05, **P < 0.01). Values of P < 0.1 were considered marginally significant. Distilled water was used as the control.

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  Figure 8. Effects of Leu‐Tyr‐Ala on lifespan in daf‐12 and daf‐16 mutants. (A) Changes in lifespan of daf‐12 (tm6046) mutants treated with 5 mmol/L Leu‐Tyr‐Ala; (B) changes in lifespan of daf‐16 (mu86) mutants treated with 5 mmol/L Leu‐Tyr‐Ala. Data are expressed as mean ± SEM. Experiments were independently conducted three times, and distilled water was used as the control. The log‐rank test was used to determine significant differences (P < 0.05).

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  Figure 9. Proposed mechanism of the anti‐aging effects of Leu‐Tyr‐Ala in Caenorhabditis elegans through acn‐1, daf‐12, and daf‐16.